Compositions and methods for treating cancer and promoting wound healing

ABSTRACT

Protein complex variants, compositions, and methods of use thereof are provided. The protein complex variant includes a cholera toxin B subunit variant having one or more modifications thereto. The method of use thereof includes treating a disease by administering an effective amount of a composition including a cholera toxin B subunit variant to a subject in need thereof.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/186,151, filed Jun. 29, 2015, and U.S. Provisional ApplicationSer. No. 62/246,367, filed Oct. 26, 2015, both of which are incorporatedherein by this reference.

GOVERNMENT INTEREST

This invention was made with government support under grant no.W81WH-10-2-0082-CLIN 2 awarded by U.S. Department of Defense. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The presently-disclosed subject matter relates to compositions andmethods for treating cancer and promoting wound healing. In particular,the presently-disclosed subject matter relates to compositions andmethods for treating cancer and promoting wound healing that make use ofa plant produced cholera-toxin B subunit (CTBp) variant.

BACKGROUND

Cholera toxin (CT), a virulence factor of Vibrio cholerae, induces anacute diarrheal response in the gut. Two major subunits make up CT, thetoxic ADP-ribosylating CTA subunit and the non-toxic,GM1-ganglioside-binding CTB subunit. The CTB subunit consists of apentameric structure with a molecular mass of approximately 55 kD and iscurrently used in World Health Organization (WHO)-prequalified oralcholera vaccines due to its capacity to induce CT-neutralizingantibodies. Additionally, CTB is often used as an adjuvant or amolecular scaffold of subunit vaccines because of its strong mucosalimmunogenicity.

It is further appreciated that CTB may induce anti-inflammatory andregulatory T cell responses and suppress immunopathological reactions inallergy and autoimmune diseases. For example, the airway administrationof CTB ameliorated experimental asthma in a murine model. In a PhaseI/II clinical trial, oral administration of CTB, chemically cross-linkedto a peptide from the human 60 kDa heat shock protein, blunted uveitisof Behcet's disease. CTB was also shown to blunt the intestinalinflammation of Crohn's disease in mice and humans. These findingsindicate the potential of CTB, in addition to its use as a choleravaccine, as an oral immunotherapeutic agent to blunt intestinalinflammation in Inflammatory Bowel Disease (IBD). However, acomprehensive investigation of CTB's effect on the gastrointestinal (GI)tract has not been done, thus leading to some debate on the protein'susefulness.

In previous studies, a non-glycosylated variant of CTB (CTBp) wasrapidly and efficiently manufactured in Nicotiana benthamiana plants.CTBp showed comparable GM1 binding affinity, physicochemical stabilityand immunogenicity to native (E. coli produced) CTB. Additionally,antibodies elicited by oral administration of CTBp in mice were able toneutralize the cholera holotoxin. These results indicated that CTBpprovides a viable alternative to the recombinant protein antigenincluded in Dukoral® oral cholera vaccines, potentially facilitatingreactive mass vaccination to respond to cholera outbreaks.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter includes protein complexvariants, compositions, and methods of use thereof. In some embodiments,the presently-disclosed subject matter relates to methods of treating adisease, comprising administering to a subject in need thereof aneffective amount of a composition including a cholera toxin B subunitvariant having one or more modifications. In some embodiments, the oneor more modification to the cholera toxin B subunit variant increasesthe expression of the polypeptide in a plant cell. In one embodiment,the modification reduces N-glycosylation. In another embodiment, themodification facilitates recombinant production of the variant. In someembodiments, the cholera toxin B subunit variant is substantiallyimmunologically identical to a cholera toxin B subunit.

Suitable modifications include, but are not limited to, sequencemodifications and/or mutations, sequence attachments, or a combinationthereof. For example, in some embodiments, the modification includes anAsn4 to Ser mutation. In some embodiments, the modification includes anattached C-terminal hexapeptide sequence. In one embodiment, theattached C-terminal hexapeptide sequence provides endoplasmic reticulum(ER) retention. In some embodiments, the cholera toxin B subunit variantcomprises the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 25.In some embodiments, the one or more modifications comprise a secretorysignal peptide selected from the group consisting of a ricealpha-amylase secretory signal peptide, a Nicotiana plumbagenifoliacalreticulin secretory signal peptide, an apple pectinase secretorysignal peptide, and a barley alpha-amylase secretory signal peptide. Insome embodiments, the one or more modifications comprise a secretorysignal peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 18, 20, 22, and 24. In some embodiments, thesecretory signal peptide comprises the rice alpha-amylase secretorysignal peptide. In some embodiments, the polypeptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NOS: 26-29.In some embodiments, the one or more modifications comprise anendoplasmic reticulum retention signal having the amino acid sequenceKDEL or HDEL. In some embodiments, the KDEL or HDEL sequence is attacheddirectly to the cholera toxin polypeptide without the use of a linkersequence. In some embodiments, the cholera toxin B subunit variantincludes two or more N-linked glycosylation sequons. For example, in oneembodiment, the cholera toxin B subunit variant comprises the amino acidsequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:14.

In some embodiments, the disease includes an inflammatory disorderand/or cancer. In one embodiment, the cancer is colon cancer. In anotherembodiment, the cancer is colitis associated colon cancer. In oneembodiment, the inflammatory disorder is an inflammatory bowel disease,such as, but not limited to, ulcerative colitis or Crohn's disease. Inanother embodiment, the inflammatory disorder is a gastrointestinalinflammation and/or injury, such as, but not limited to, celiac disease,irritable bowel syndrome, radiation-induced colitis, orinfection-induced colitis. In another embodiment, the inflammatorydisorder is a mucosal inflammation and/or injury, such as, but notlimited to, asthma, airway burns, corneal injury, or vaginosis.

The method of treating a disease includes administering the compositionby any suitable route. For example, in one embodiment, the methodincludes oral administration of the composition. In some embodiments,the method includes administering the composition to the subject withoutsubstantially changing a fecal microbiome of the subject. In someembodiments, administering the composition decreases protein levels oftumor promoting cytokines. In some embodiments, administering thecomposition increases the innate immune cell populations in thesubject's colon. In one embodiment, administering the compositionincreases the innate immune cell populations without producingsubstantial effect on adaptive immune cell populations. In someembodiments, administering the composition provides an increased effecton colon gene expression as compared to small intestine gene expression.

In some embodiments, the presently-disclosed subject matter relates tomethods of enhancing wound healing, comprising administering to asubject in need thereof an effective amount of a composition including acholera toxin B subunit variant having one or more modifications. Insome embodiments, the wound healing comprises mucosal wound healing. Insome embodiments, the one or more modification to the cholera toxin Bsubunit variant increases the expression of the polypeptide in a plantcell. In one embodiment, the modification reduces N-glycosylation. Inanother embodiment, the modification facilitates recombinant productionof the variant. Suitable modifications include, but are not limited to,an Asn4 to Ser mutation, an attached C-terminal hexapeptide sequence, ora combination thereof. In one embodiment, the modification providesendoplasmic reticulum (ER) retention. In some embodiments, the choleratoxin B subunit variant is substantially immunologically identical to acholera toxin B subunit.

In some embodiments, the presently-disclosed subject matter relates tocholera toxin B subunit variants. In some embodiments, the cholera toxinB subunit variants are immunologically identical to natural choleratoxin B subunits. In some embodiments, the cholera toxin B subunitvariants include one or more modification to increase the expression ofthe polypeptide in a plant cell, reduce N-glycosylation, and/orfacilitate recombinant production. In some embodiments, the choleratoxin B subunit variants are produced by recombinant production inplants, E. coli, yeast, insect cells, mammalian cells, or a combinationthereof.

Suitable modifications of the cholera toxin B subunit variants include,but are not limited to, sequence modifications and/or mutations,sequence attachments, or a combination thereof. For example, in someembodiments, the modification includes an Asn4 to Ser mutation. In someembodiments, the modification includes an attached C-terminalhexapeptide sequence. In one embodiment, the attached C-terminalhexapeptide sequence provides endoplasmic reticulum (ER) retention. Insome embodiments, the cholera toxin B subunit variant comprises thesequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 25. In someembodiments, the one or more modifications comprise a secretory signalpeptide selected from the group consisting of a rice alpha-amylasesecretory signal peptide, a Nicotiana plumbagenifolia calreticulinsecretory signal peptide, an apple pectinase secretory signal peptide,and a barley alpha-amylase secretory signal peptide. In someembodiments, the one or more modifications comprise a secretory signalpeptide having an amino acid sequence selected from the group consistingof SEQ ID NOS: 18, 20, 22, and 24. In some embodiments, the secretorysignal peptide comprises the rice alpha-amylase secretory signalpeptide. In some embodiments, the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 26-29. Insome embodiments, the one or more modifications comprise an endoplasmicreticulum retention signal having the amino acid sequence KDEL or HDEL.In some embodiments, the cholera toxin B subunit variant includes two ormore N-linked glycosylation sequons. For example, in one embodiment, thecholera toxin B subunit variant comprises the amino acid sequence of SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

Further features and advantages of the present invention will becomeevident to those of ordinary skill in the art after a study of thedescription, figures, and non-limiting examples in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes graphs showing results of flow cytometry performed oneweek after CTBp administration.

FIG. 2 includes graphs showing results of flow cytometry performed twoweeks after CTBp administration.

FIG. 3 includes graphs showing flow cytometry analysis of the colonlamina propria immune cell population.

FIG. 4A is a heat map showing microarray analysis of gene expression inthe colon epithelia.

FIG. 4B is a transcriptome profile of intestinal genes after CTBp oraladministration.

FIG. 4C is a diagram describing pathways affected by CTBp oraladministration.

FIG. 5 is a schematic diagram showing a microarray TGFb pathwayanalysis.

FIG. 6A includes graphs illustrating qPCR analysis of collagen, decorin,mitogen-activated protein KKKK4, and tissue inhibitor ofmetalloproteinase 4.

FIG. 6B includes graphs illustrating qPCR analysis of malatedehydrogenase 1 and angiogenin.

FIG. 6C includes graphs illustrating qPCR analysis of ATP bindingcassette and SMAD family member 6.

FIGS. 7A-7C include schematic diagrams and graphs showing gut microbiomeanalysis of mice administered with CTBp or PBS at the phylum and specieslevels. Fecal samples were collected from each mouse 2 weeks after thesecond dose of PBS or CTBp. Following bacterial DNA isolation sampleswere analyzed. (FIG. 7A) A representative sample of the phylum levelabundance analysis. (FIG. 7B) Representative samples of the specieslevel abundance analysis and top 6 represented species in the samples.(FIG. 7C) Operational Taxonomic Units (OTUs) representing the lowestp-values as determined by ANOVA. CTBp did not significantly affect theoverall composition of the gut microbiota, but the abundance of severalminor species in the Firmicutes phylum was significantly altered.

FIG. 8A includes photomicrographs of wounded CaCo2 cells, where the invitro wound closure was recorded over 24 hours and microscopicallyanalyzed.

FIG. 8B is a graph illustrating analysis of in vitro wound closure after24 hours by wound area measurement. Graphs represent percent woundclosure after 24 hour (*, ** ***=p<0.05, 0.01, 0.001; One-way ANOVA withBonferroni's multiple comparison tests).

FIG. 8C is a graph showing that the enhancement of wound closureprovided by TGFβ or 1 μM CTBp was inhibited by anti-TFGβ antibody.Graphs represent percent wound closure after 24 hour (*, **, ***=p<0.05,0.01, 0.001; One-way ANOVA with Bonferroni's multiple comparison tests).

FIGS. 9A-9E include graphs and images illustrating CTBp's effects in anacute DSS colitis model one week after DSS exposure. (FIG. 9A) A graphillustrating weight loss as percent change body weight for miceadministered PBS, PBS+DSS, and CTBp+DSS. (FIG. 9B) A graph illustratinginflammation score for mice administered PBS, PBS+DSS, and CTBp+DSS.(FIG. 9C) Representative photomicrographs of tissue from miceadministered PBS, PBS+DSS, and CTBp+DSS. (FIG. 9D) Graphs illustratingvarious gene expression levels for mice administered PBS, PBS+DSS, andCTBp+DSS. (FIG. 9E) Graphs illustrating various protein levels for miceadministered PBS, PBS+DSS, and CTBp+DSS.

FIGS. 10A-10C are graphs and images illustrating CTBp's effects in anacute DSS colitis model immediately after DSS exposure. (FIG. 10A)Representative photomicrographs of tissue from mice administered PBS,CTBp, PBS+DSS, MES+DSS, and CTBp+DSS. (FIG. 10B) A graph illustratinginflammation score for mice administered PBS, CTBp, PBS+DSS, MES+DSS,and CTBp+DSS. (FIG. 10C) Graphs illustrating various gene expressionlevels for mice administered PBS, PBS+DSS, and CTBp+DSS. (FIG. 10D)Graphs illustrating various protein levels for mice administered PBS,PBS+DSS, and CTBp+DSS.

FIGS. 11A-11D are graphs and images illustrating CTBp's effects in achronic colitis/colon cancer model. (FIG. 11A) A graph showing diseaseactivity index for mice administered various different compositions. Thegraph illustrates CTBp's effects on disease activity index in a chroniccolitis/colon cancer model. (FIG. 11B) Representative images of varioustumor grades. (FIG. 11C) A graph showing tumor numbers for miceadministered various different compositions. The graph illustratesCTBp's effects on tumor number in a chronic colitis/colon cancer model.(FIG. 11D) A graph showing total tumor score for mice administeredvarious different compositions. The graph illustrates CTBp's effects ontumor score in a chronic colitis/colon cancer model.

FIG. 12 is a schematic diagram of the experimental design according toan embodiment of the disclosure.

FIG. 13A is a graph illustrating percent body weight change in acuteulcerative colitis.

FIG. 13B is a graph illustrating colon inflammation scoring in acuteulcerative colitis.

FIG. 13C includes representative composite photomicrographs depictingPBS, PBS+DSS, and CTBp+DSS.

FIG. 14 is a graph illustrating disease activity index (DAI). Two wayANOVA was performed b=p<0.05 compared to PBS+AOM+DSS.

FIG. 15A is a graph showing average number of tumors per mouse.

FIG. 15B is a graph showing the number of tumors per grade level.

FIG. 15C is a graph showing total tumor score per mouse in relation totumor number. ANOVA was performed, p<0.05 compared to PBS.

FIG. 16 includes representative photomicrographs of various tumorgrades.

FIG. 17 includes graphs showing how CTBp significantly altered geneexpression. ANOVA was performed a=p<0.05 compared to PBS and b=p<0.05compared to PBS+AOM+DSS.

FIG. 18 includes graphs showing how CTBp significantly decreasedinflammatory protein levels. ANOVA was performed a=p<0.05 compared toPBS and b=p, 0.05 compared to PBS+AOM+DSS.

FIG. 19 includes a schematic diagram and graphs showing experimentaldesign, disease activity index, and inflammation score at various timepoints according to an embodiment of the disclosure. *P<0.05 compared toPBS-DSS, One-way ANOVA with Bonferroni's multiple comparison test.

FIG. 20 includes a schematic diagram and graphs showing experimentaldesign, disease activity index, colon length, inflammation score atvarious dosage levels according to an embodiment of the disclosure. *,**, ***P<0.05, 0.01, 0.001 compared to PBS-DSS, One-way ANOVA withBonferroni's multiple comparison tests.

FIG. 21 includes graphs showing cell levels in small intestine laminapropria.

FIG. 22 includes graphs showing cell levels in colon lamina propria.

FIG. 23 includes graphs illustrating how extracellular matrix componentsare significantly increased by CTBp administration.

FIG. 24 includes graphs illustrating how remodeling enzymes aresignificantly increased by CTBp administration.

FIG. 25 includes graphs illustrating gene expression data and tissueprotein levels. *, **, ***P<0.05, 0.01, 0.001 compared to PBS-DSS,One-way ANOVA with Bonferroni's multiple comparison tests

FIG. 26 is a graph illustrating the number of colon tumors per mouse.a=p<0.05 (vs PBS); b=p<0.05 (vs PBS+AOM/DSS), one-way ANOVA withNewman-Keuls Multiple Comparison Test.

FIG. 27 includes representative images illustrating wound healing over aperiod of 48 hours for CaCO₂. *, **, ***P<0.05, 0.01, 0.001 compared toPBS; images were taken at ×4 magnification.

FIG. 28 is a graph illustrating percent wound closure.

FIG. 29 is a graph comparing percent change body weight of PBS and CTBp.

FIG. 30 is a graph comparing percent change body weight of PBS, PBS+DSS,and 30 μg CTBp+DSS. a=p<0.05 compared to PBS. b=p<0.05 compared toPBS+DSS.

FIG. 31 includes representative images of hematoxylin and eosinstaining.

FIG. 32 includes representative images of hematoxylin and eosinstaining.

FIG. 33 includes representative images of trichrome stain.

FIG. 34 is a graph illustrating inflammation scoring. a=p<0.05 comparedto PBS; b=p<0.05 compared to PBS+DSS.

FIG. 35 is a schematic illustration of an experimental design accordingto an embodiment of the disclosure.

FIG. 36 is a graph illustrating disease activity index of PBS, CTBp, andCTB. **P<0.01; one-way ANOVA with Bonferroni's multiple comparisontests.

FIGS. 37A-37C includes graphs and images showing CTBp significantlyalters the immune cell profile in the colon. Animals were orallyadministered PBS or CTBp twice, at a two-week interval and two weekslater the mice were sacrificed. Colon lamina propria leukocytes wereisolated and stained for surface and internal markers specific forimmune cell subtypes. CD4⁺ and CD8⁺ cells gated on T lymphocytesubpopulation (CD45⁺CD3⁺). Additionally, CD45⁺ cells were furtherdivided into B (CD19⁺), macrophage (F4/80⁺), dendritic (CD11c⁺) andnatural killer (CD49b⁺) subpopulations. Dot plots are representativesamples from each group. Data presented as mean±standard error of themean (SEM) of at least four biological replicates comprised of twopooled mice each. Unpaired t test was performed with *P<0.05 compared toPBS group. (FIG. 37A) Innate immune cell populations in the colon laminapropria. (FIG. 37B) Adaptive immune cell populations in the colon laminapropria. (FIG. 37C) Immunohistochemistry analysis of macrophage (F4/80⁺)cells in the distal colon lamina propria isolated from mice 2 weeks postsecond CTBp oral administration. Paraffin embedded colon sections wereincubated with F4/80 primary antibody (1:100 dilution) and abiotinylated secondary antibody. After addition of a horseradishperoxidase (HRP) and 3,3′-diaminobenzidine tetrahydrochloride (DAB)solution, positive cells were counted in 10 high power fields persection and averaged for each colon. Mean±SEM is shown. Unpaired t testwas performed with *P<0.05 compared to PBS group. Animals per group: PBS(n=6) and 30 μg CTBp (n=7).

FIGS. 38A-38C include graphs and schematic diagrams showingTGFβ-dependent pathways are significantly altered by CTBp in the colon.PBS or CTBp was administered twice to mice at a two week interval. Twoweeks after the final dose animals were sacrificed and the smallintestine and colon were removed for RNA purification. Total RNA wasamplified and labeled, and then whole transcript expression analysis wasperformed. (FIG. 38A) Heat map showing differentially expressed genes inthe small intestine (SI) and colon (COL) following PBS or CTBpadministration. (FIG. 38B) Number of significantly altered genes in thecolon and small intestine following PBS or CTBp administration.Significance was determined at P<0.01.

(FIG. 38C) Ten most significantly enhanced and suppressed pathways byCTBp administration in the colon as determined by MetaCore™ ontologiesenrichment analysis using P<0.01 and a fold change of <−1.2 or >1.2.

FIGS. 39A-39D includes graphs and images showing that CTBp enhanceswound healing in a human colon epithelial model. Caco2 cells were grownto confluence and scratched with a pipette tip. Cells were thenincubated with PBS, TGFβ1, anti-TGFβ1,2,3 antibody, and/or CTBp. The invitro wound closure was recorded over 48 h and 4× magnification imageswere acquired with a EVOS® fl by Advanced Microscopy Group and meanpercentage closure was determined by Image J software. (FIG. 39A)Photomicrographs of wounded Caco2 cells. (FIG. 39B) Analysis of in vitrowound closure after 24 and 48 h by wound area measurement. Means±SEM offour independent experiments are shown. **P<0.01, and ***P<0.001;one-way ANOVA with Bonferroni's multiple comparison tests. (FIG. 39C)Analysis of in vitro wound closure after 24 h by wound area measurementafter incubation with PBS or an anti-TGFβ1,2,3 antibody coincubated with1.0 μM CTBp or 0.2 nM TGFβ1. Means±SEM of four independent experimentsare shown. *P<0.05, **P<0.01, and ***P<0.001; one-way ANOVA withBonferroni's multiple comparison tests. (d) Protein concentrations inCaco2 cell supernatants. Means±SEM of four independent experiments areshown. *P<0.05, **P<0.01, and ***P<0.001; one-way ANOVA withBonferroni's multiple comparison tests.

FIG. 40A-40G includes graphs and images showing that oral administrationof CTBp blunts DSS-induced colonic epithelial damage. Mice were orallyadministered PBS or CTBp twice at a two-week interval. Immediately afterthe second administration DSS exposure began and continued for 8 days.Colon tissues were isolated after a 6-day recovery for analyses.Mean±SEM is shown for each group. Animals per group: PBS (n=6), 30 μgCTBp+DSS=(n=8), and PBS+DSS (n=8). (FIG. 40A) Percent change of bodyweights. Animals were weighed daily and just prior to the initiation ofDSS exposure. Percent change was based on the initial body weight.**P<0.01 between DSS-exposed, CTBp- and PBS-administered groups; two-wayANOVA with Bonferroni's multiple comparison tests. (FIG. 40B) Coloninflammation scoring. Paraffin embedded tissue sections were scoredafter staining with Hematoxylin and Eosin (H&E). Scoring was based on a0 to 4 scale. (FIG. 40C) Representative 4× (left) and 20× (right)photomicrographs of colons from treatment groups. (FIGS. 40D-40E)Immunohistochemistry staining of F4/80⁺ cells in distal colon tissue.Means±SEM of positive cells from 10 individual microscope fields persample (FIG. 40D) and representative photographs (FIG. 40E) are shown.(FIG. 40F) Representative photomicrographs of colons following Masson'sTrichrome Stain. (FIG. 40G) qRT-PCR analysis of cytokine gene expressionin mouse colon tissue. Mean±SEM is shown for each group (N=5). One-wayANOVA with Bonferroni's multiple comparison test was used for (b, d, g).*P<0.05, **P<0.01, ***P<0.001.

FIGS. 41A-41C include graphs and images showing that CTBp significantlyreduces colon inflammation induced by DSS exposure. Mice were orallyadministered with PBS or CTBp and exposed to DSS as in FIG. 40. As areference control, a group of mice were treated with oral administrationof 100 μg mesalamine daily during the DSS exposure. Colon tissues wereisolated immediately after the DSS exposure for analyses. (FIG. 41A)Representative photomicrographs of colons from the treatment groups andcolon inflammation scoring. Paraffin embedded tissue sections werescored after staining with H&E. Scoring was based on a 0 to 4 scale.Mean±SEM is shown for each group. Animals per group: PBS (n=8), 30 μgCTBp+DSS (n=8), MES+DSS (n=9) and PBS+DSS (n=7). (FIG. 41B) qRT-PCRanalysis of cytokine gene expression in mouse colon tissue. Mean±SEM isshown for each group (N=5). (FIG. 41C) Cytokine concentrations in colontissue lysate. N=5 per group. One-way ANOVA with Bonferroni'smultiple-comparison post-test (b) or Kruskal-Wallis test with Dunn'smultiple-comparison post-test were used to compare groups. *P<0.05,**P<0.01 and ***P<0.001.

FIGS. 42A-42D include graphs and images showing the therapeutic dosingof CTBp is effective in ameliorating DSS-induced acute colitis. Micewere exposed to 3% DSS for seven days and orally administered with PBSor CTBp on the third and sixth day. Colon tissues were isolated after atwo-day recovery for analyses. N=5 for PBS and N=10 for all othergroups. (FIG. 42A) Disease activity index (DAI) scores. Body weightloss, fecal consistency and occult blood were scored at the time ofsacrifice. (FIG. 42B) Colon length. (FIG. 42C) Colon inflammationscoring. Paraffin embedded tissue sections were scored after stainingwith H&E. Scoring was based on a 0 to 4 scale. Mean±SEM is shown foreach group. *P<0.05, **P<0.01 and ***P<0.001; one-way ANOVA withBonferroni's multiple comparison tests. (d) Representative 4× (top) and20× (bottom) photomicrographs of H&E-stained distal colon tissues fromeach group.

FIGS. 43A-43F include graphs and images showing biweekly oral therapywith CTBp can mitigate chronic colitis and colitis-associatedtumorigenesis. Mice received azoxymethane (AOM) i.p. one week prior toDSS exposure. Mice were given water or 2% DSS water for 7 days followedby water for two weeks; the cycle was repeated two additional times.Mice were dosed orally with PBS, 3 μg CTBp or 10 μg CTBp on days 7, 21,35, and 49 (black arrows). (FIG. 43A) DAI. Body weights, fecalconsistency and occult blood were scored daily. *P<0.05 compared toPBS+DSS group; a repeated-measures ANOVA with Bonferroni Correction.(FIG. 43B) Representative tumor scoring. Tumors were scored from 0 to 5with 0 being normal tissue and 5 being greater than 50% of coloncircumference tumor invasion. Red arrows indicate representative tumors.(FIG. 43C) Tumor scoring results. Tumors were scored based on the scalein b. ^(##)P<0.01 and ^(###)P<0.001 compared to PBS and **P<0.01compared to PBS+DSS; a repeated-measures ANOVA with BonferroniCorrection. (FIG. 43D) Tumor score vs tumor number. Total tumor numberis the X axis and total tumor grade is the Y axis. A dot represent eachmouse. Mice with no tumors are at the axis intersection. Mean±SEM isshown (n=5 for PBS group and all other groups n=10). (FIG. 43E) qRT-PCRanalysis of cytokine gene expression in mouse colon tissue. Mean±SEM isshown for each group (N=5). (FIG. 43F) Cytokine concentrations in colontissue lysate. N=5 per group. For e and f, a Kruskal-Wallis test withDunn's multiple-comparison post-test was used. *P<0.05 and **P<0.01.

FIG. 44 includes graphs showing immune cell populations in differentlymphoid tissues two weeks after the second CTBp or PBS oraladministration. Animals were orally administered twice at a two-weekinterval with PBS or CTBp and two weeks later the mice were sacrificed.Small intestine, Peyer's patches, and spleen lymphocytes were isolatedafter several wash steps and collagenase incubation steps as necessary.CD4⁺ and CD8⁺ cells gated on gated on T lymphocyte subpopulation(CD45⁺CD3⁺). Additionally, CD45⁺ cells were further divided into B(CD19⁺), macrophage (F4/80⁻), dendritic (CD11c⁻) and natural killer(CD49b⁺) subpopulations. Dot plots are representative samples taken fromeach group. Data are presented as mean±standard error of the mean (SEM)of at least four biological replicates comprised of two pooled miceeach. Unpaired t test was performed with *P<0.05 compared to PBS group.Colon lamina propria immune cell profiles are shown in FIG. 1.

FIG. 45 is a graph showing a principal coordinate analysis that revealedseparation of GI tract gene expression profiles of mice vaccinated withPBS and CTBp. PBS or CTBp were administered twice to mice at a two-weekinterval. Two weeks after the final dose animals were sacrificed and thesmall intestine and colon was removed. RNA was purified from smallintestine and colon tissue sections. Total RNA was amplified and labeledthen whole transcript expression analysis was performed as described inMaterials and Methods. Principal coordinate analysis was performed usingPartek Genomics Suite 6.6 (St. Louis, Mo.). N=3 for all groups.

FIG. 46 is a schematic diagram showing TGFβ-associated gene expressionpathways in the colon induced by CTBp oral administration. Pathwayanalysis of colon gene expression from microarray analysis is shown. Redbars are indicative of significant induction of the gene. Pathwayanalysis was performed by MetaCore™ ontologies enrichment analysis usingP<0.01 (one-way ANOVA) and a fold change of greater or less than 1.2.Definitions: EMT=epithelial to mesenchymal transition,MAPK=mitogen-activated protein kinase, HGF=hepatocyte growth factor.

FIGS. 47A-47C includes graphs showing qRT-PCR analysis of representativegenes that were shown to be induced by CTBp administration in microarrayanalysis. RNA samples from the distal colon tissue were isolated usingthe Qiagen RNeasy Microarray Tissue Mini Kit and analyzed by AppliedBiosystems TaqMan Array 96-Well FAST plate in an Applied Biosystems 7500Fast Real-Time PCR System. N=3 per group. *P<0.05, **P<0.01 and***P<0.001; unpaired t test. (FIG. 47A) Microarray-identifiedsignificantly induced genes including: Col1a1, Dcn, Map4k4 and Timp4.(FIG. 47B) Microarray identified suppressed genes including: Mdh1 andAng4. (FIG. 47C) Unchanged genes in microarray analysis including: Abca1and Smad6.

FIGS. 48A-48D are graphs showing wound healing pathway-focused qRT-PCRanalysis of colon gene expression. Two weeks post administration, RNAsamples were isolated using the Qiagen RNeasy Microarray Tissue Mini Kitand analyzed by RT2 Profiler PCR Mouse Wound Healing Array (Qiagen, Cat.No. PAMM-121Z) in an Applied Biosystems 7500 Fast Real-Time PCR System.N=3 per group. A 1.5 fold change from PBS cutoff was used to filter dataprior to testing for significance. Seven significantly changed genes in:(FIG. 48A) extracellular matrix structural constituents; (FIG. 48B)extracellular matrix remodeling enzymes; (FIG. 48C) cytoskeletonregulators; and (FIG. 48D) growth factors, are shown. *P<0.05 and**P<0.01; unpaired t tests.

FIG. 49 is a graph showing that CTBp enhances TGFβ mediated woundhealing in a human colon epithelial model. Caco2 cells were grown toconfluence and scratched with a pipette tip. Cells were then incubatedwith PBS, TGFβ, Anti-TGFβ, and/or CTBp. The in vitro TGFβ levels weremeasured after 24 by a Luminex Multiplex assay. Mean±SEM is shown (N=4,experimental replicates per group). *P<0.05, **P<0.01, and ***P<0.001;one-way ANOVA with Bonferroni's multiple comparison tests.

FIG. 50 is a schematic diagram showing study design for acute colitisexperiments. Mice were orally administered PBS or CTBp twice at atwo-week interval. Immediately after the second administration DSSexposure began for 8 days. Animals were sacrificed either immediatelyafter the DSS exposure period or after a 6 day recovery.

FIG. 51 is a schematic diagram showing chronic colitis study design.Animals in the indicated groups were injected i.p. with 10 mg/kgazoxymethane (AOM). DSS exposure began 1 week after AOM injection andcontinued for 1 week. Immediately after removal of DSS water, PBS orCTBp (3 or 10 μg per mouse) was administered to the respective groupsand dosing was continued biweekly for a total of 4 doses. Animals wereallowed to recover for 2 weeks prior to the beginning of a second DSSexposure period. The DSS exposure/2 week recovery cycle was repeated 2additional times for a total of 3 cycles. Animals were sacrificedfollowing the final 2 week recovery.

FIG. 52 is a graph showing principal coordinate analysis of gutmicrobiome two weeks following the second CTBp or PBS administration.Fecal samples were collected from each mouse 2 weeks after the finaldose of PBS or CTBp. Following bacterial DNA isolation samples wereanalyzed. No significant alteration of overall microbiome was notedfollowing CTBp administration. Weighted UniFrac metric was performed on1,896 taxa. No significant change in overall microbiome was observedbetween the two groups.

FIGS. 53A-53C include schematic diagrams and graphs showing gutmicrobiome analysis of mice administered with CTBp or PBS at the phylumand species levels. Fecal samples were collected from each mouse 2 weeksafter the second dose of PBS or CTBp. Following bacterial DNA isolationsamples were analyzed. (FIG. 53A) A representative sample of the phylumlevel abundance analysis. (FIG. 53B) Representative samples of thespecies level abundance analysis and top 6 represented species in thesamples. (FIG. 53C) Operational Taxonomic Units (OTUs) representing thelowest p-values as determined by ANOVA. CTBp did not significantlyaffect the overall composition of the gut microbiota, but the abundanceof several minor species in the Firmicutes phylum was significantlyaltered.

FIGS. 54A-54C are graphs showing a comparison of plant-producedN4S-CTB-SEKDEL (CTBp) with original CTB in an acute colitis mouse model.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is nucleic acid sequence of a wild-type cholera toxin Bsubunit from Vibrio cholerae;

SEQ ID NO: 2 is an amino acid sequence of a wild-type cholera toxin Bsubunit from Vibrio cholerae;

SEQ ID NO: 3 is nucleic acid sequence encoding a cholera toxin B subunitvariant polypeptide modified to include a C-terminal endoplasmicreticulum signal and to include no N-linked glycosylation sequons atAsn4;

SEQ ID NO: 4 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide modified to include a C-terminal endoplasmicreticulum signal and to include no N-linked glycosylation sequons atAsn4;

SEQ ID NO: 5 is a nucleic acid sequence encoding a cholera toxin Bsubunit variant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and having one N-linked glycosylation sequonat Asn4;

SEQ ID NO: 6 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and having one N-linked glycosylation sequonat Asn4;

SEQ ID NO: 7 is a nucleic acid sequence encoding a cholera toxin Bsubunit variant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include two N-linked glycosylationsequons at Asn4 and Asn103;

SEQ ID NO: 8 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include two N-linked glycosylationsequons at Asn4 and Asn103;

SEQ ID NO: 9 is a nucleic acid sequence encoding another cholera toxin Bsubunit variant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include two N-linked glycosylationsequons at Asn4 and Asn21;

SEQ ID NO: 10 is an amino acid sequence of another cholera toxin Bsubunit variant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include two N-linked glycosylationsequons at Asn4 and Asn21;

SEQ ID NO: 11 is a nucleic acid sequence encoding a cholera toxin Bsubunit variant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include three N-linked glycosylationsequons at Asn4, Asn21, and Asn103;

SEQ ID NO: 12 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include three N-linked glycosylationsequons at Asn4, Asn21, and Asn103;

SEQ ID NO: 13 is a nucleic acid sequence encoding another cholera toxinB subunit variant polypeptide modified to include a C-terminalendoplasmic reticulum retention signal and to include three N-linkedglycosylation sequons at Asn4, Asn21, and Asn103;

SEQ ID NO: 14 is an amino acid sequence of another cholera toxin Bsubunit variant polypeptide modified to include a C-terminal endoplasmicreticulum retention signal and to include three N-linked glycosylationsequons at Asn4, Asn21, and Asn103;

SEQ ID NO: 15 is a nucleic acid sequence encoding a cholera toxin Bsubunit variant polypeptide with an N-terminal secretory signal fromVibrio cholerae and a C-terminal endoplasmic reticulum retention signal;

SEQ ID NO: 16 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide including an N-terminal secretory signal from Vibriocholerae and a C-terminal endoplasmic reticulum retention signal;

SEQ ID NO: 17 is a nucleic acid sequence encoding a rice alpha-amylasesecretory signal peptide;

SEQ ID NO: 18 is an amino acid sequence of a rice alpha-amylasesecretory signal peptide;

SEQ ID NO: 19 is nucleic acid sequence encoding a Nicotianaplumbagenifolia calreticulin secretory signal peptide;

SEQ ID NO: 20 is an amino acid sequence of a Nicotiana plumbagenifoliacalreticulin secretory signal peptide;

SEQ ID NO: 21 is a nucleic acid sequence encoding an apple pectinasesecretory signal peptide;

SEQ ID NO: 22 is an amino acid sequence of an apple pectinase secretorysignal peptide;

SEQ ID NO: 23 is a nucleic acid sequence encoding a barley alpha-amylasesecretory signal peptide;

SEQ ID NO: 24 is an amino acid sequence encoding a barley alpha-amylasesecretory signal peptide;

SEQ ID NO: 25 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide including a Ser26→Cys and an Ala102→Cys mutation;

SEQ ID NO: 26 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide including a rice alpha-amylase N-terminal secretorysignal peptide and a C-terminal endoplasmic reticulum retention signalpeptide;

SEQ ID NO: 27 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide including a Nicotiana plumbagenifolia calreticulinN-terminal secretory signal peptide and a C-terminal endoplasmicreticulum retention signal peptide;

SEQ ID NO: 28 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide including an apple pectinase N-terminal secretorysignal peptide and a C-terminal endoplasmic reticulum retention signalpeptide;

SEQ ID NO: 29 is an amino acid sequence of a cholera toxin B subunitvariant polypeptide including a barley alpha-amylase N-terminalsecretory signal peptide and a C-terminal endoplasmic reticulumretention signal peptide;

SEQ ID NO: 30 is an amino acid sequence of an exemplary endoplasmicreticulum retention signal peptide, KDEL, including a two amino acidlinker, SE, preceding the KDEL sequence; and

SEQ ID NO: 31 is an amino acid of the exemplary endoplasmic reticulumretention signal peptide, KDEL.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

Some of the polynucleotide and polypeptide sequences disclosed hereinare cross-referenced to GENBANK®/GENPEPT® accession numbers. Thesequences cross-referenced in the GENBANK®/GENPEPT® database areexpressly incorporated by reference as are equivalent and relatedsequences present in GENBANK®/GENPEPT® or other public databases. Alsoexpressly incorporated herein by reference are all annotations presentin the GENBANK®/GENPEPT® database associated with the sequencesdisclosed herein. Unless otherwise indicated or apparent, the referencesto the GENBANK®/GENPEPT® database are references to the most recentversion of the database as of the filing date of this Application.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GenBank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Cholera toxin B subunit (CTB) is a pleiotropic mucosal immunomodulatoryprotein and currently used as an active component of an internationallylicensed oral cholera vaccine. However, CTB's immunomodulatory effect onthe gastrointestinal tract has not been fully investigated. To that end,the presently-disclosed subject matter is based, at least in part, onthe discovery that oral administration of a CTB variant (CTBp)significantly altered the gene expression profile in the distal colon inmice while comparatively less change was noted in the small intestine.The alterations in gene expression indicated the activation ofTGFβ-mediated pathways involved in mucosal epithelial integrity.Additionally, immune cell populations in the colon lamina propria weresignificantly altered by CTBp, whereby macrophages, dendritic cells, andnatural killer cells were increased, while B cells were decreased.Meanwhile, the fecal microbiome remained largely unchanged upon CTBporal administration, indicating that the changes in colonic geneexpression and immune cell populations are not mediated by gutmicroflora.

Given that CTBp induced TGFβ-mediated pathways and increased phagocyticinnate immune cells in the colonic mucosa, and without wishing to bebound by any particular theory, it was believed that CTBp could enhancemucosal wound healing in the colon. That belief was, at least in part,then demonstrated in an in vitro mucosal wound healing model employingCaco2 cells, in which CTBp increased wound healing in a TGFβ-dependentmanner. Interestingly, CTBp exhibited more effective wound healingactivity than native CTB. Again, without wishing to be bound by theory,the increased wound healing provided by CTBp as compared to native CTBis believed to be due to the amino acid sequence modification introducedto the former resulting in prolonged retention in the epithelial cells.CTBp's wound healing capacity was further demonstrated in a dextransodium sulfate (DSS) acute colitis model in mice. CTBp protected againstepithelial damage as manifested by mitigating body weight loss,decreasing pathological symptoms in the colon epithelia, and bluntingthe escalation of inflammatory cytokine levels. Notably, biweekly oraladministration of CTBp significantly reduced tumorigenesis in theazoxymethane/DSS model of colon cancer. Together, the resultsdemonstrated CTBp's ability to enhance mucosal wound healing, andhighlighted its potential for application in ulcerative colitis oralimmunotherapy.

The presently-disclosed subject matter thus includes compositions andmethods for treating cancer, enhancing wound healing, and/or treating aninflammatory disorder. In some embodiments, a method of treating acancer is provided, the method including administering to a subject inneed thereof an effective amount of a composition including a choleratoxin B subunit variant. In some embodiments, the cholera toxin Bsubunit variant includes one or more modifications as compared to anative cholera toxin B subunit. Such variants are referred to herein asa CTB variant (CTBp) or a cholera toxin B subunit variant polypeptide.

The term “cancer” refers to all types of cancer or neoplasm or malignanttumors found in a subject, including leukemias, lymphomas, myelomas,carcinomas, melanomas, teratomas, and sarcomas. Examples of cancersinclude cancer of the liver, pancreas, esophagus, brain, bladder,breast, central nervous system (e.g., spine), cervix, colon, rectum,head and neck, kidney, lung, ovary, prostate, sarcoma, stomach, uterus,leukemias, lymphomas, myelomas, and melanomas. In one embodiment, thecancer includes colon cancer. In another embodiment, the colon cancerincludes colitis-associated colon cancer.

In some embodiments, a method of enhancing wound healing is provided,the method including administering to a subject in need thereof aneffective amount of a composition including a cholera toxin B subunitvariant. In one embodiment, the enhanced wound healing includes enhancedmucosal wound healing. As used herein, the term “mucosal wound” refersto any injury to tissue that includes a mucosal membrane and that may becapable of producing mucus, such as, but not limited to, digestivetissue, genital tissue, and/or urinary tract tissue.

In some embodiments, a method of treating an inflammatory disorder isprovided, the method including administering to a subject in needthereof an effective amount of a composition including a cholera toxin Bsubunit variant. The term “inflammatory disorder” includes diseases ordisorders which are caused, at least in part, or exacerbated, byinflammation, which is generally characterized by increased blood flow,edema, activation of immune cells (e.g., proliferation, cytokineproduction, or enhanced phagocytosis), heat, redness, swelling, painand/or loss of function in the affected tissue or organ. The cause ofinflammation can be due to physical damage, chemical substances,micro-organisms, tissue necrosis, cancer, or other agents or conditions.

Inflammatory disorders include acute inflammatory disorders, chronicinflammatory disorders, and recurrent inflammatory disorders. Acuteinflammatory disorders are generally of relatively short duration, andlast for from about a few minutes to about one to two days, althoughthey can last several weeks. Characteristics of acute inflammatorydisorders include increased blood flow, exudation of fluid and plasmaproteins (edema) and emigration of leukocytes, such as neutrophils.Chronic inflammatory disorders, generally, are of longer duration, e.g.,weeks to months to years or longer, and are associated histologicallywith the presence of lymphocytes and macrophages and with proliferationof blood vessels and connective tissue. Recurrent inflammatory disordersinclude disorders which recur after a period of time or which haveperiodic episodes. Some inflammatory disorders fall within one or morecategories.

Exemplary inflammatory disorders include, but are not limited toatherosclerosis; arthritis; asthma; autoimmune uveitis; adoptive immuneresponse; dermatitis; multiple sclerosis; diabetic complications;osteoporosis; Alzheimer's disease; cerebral malaria; hemorrhagic fever;autoimmune disorders; and inflammatory bowel disease. In embodiments,the term “inflammatory disorder” is further inclusive ofinflammation-promoted cancers, such that the term “inflammatorydisorder” can be used to refer to cancers caused or promoted byinflammation, such as colon cancer. In some embodiments, theinflammatory disorder is selected from the group consisting of sepsis,septic shock, colitis, colon cancer, and arthritis. For example, in oneembodiment, the method of treating an inflammatory disorder includestreating an inflammatory bowel disease, such as, but not limited to,ulcerative colitis and/or Crohn's disease; a gastrointestinalinflammation and/or injury, such as, but not limited to, celiac disease,irritable bowel syndrome, radiation-induced colitis, and/orinfection-induced colitis; a mucosal inflammation and/or injury, suchas, but not limited to, asthma, airway burns, corneal injury, and/orvaginosis; or a combination thereof. The term “colitis” refers to aninflammation of the colon which may be acute or chronic.

As used herein, the terms “treatment” or “treating” relate to anytreatment of a disease of a subject, including, but not limited to,prophylactic treatment and therapeutic treatment. As such, the termstreatment or treating include, but are not limited to: preventing adisease or the development of a disease; inhibiting the progression of adisease; arresting or preventing the development of a disease; reducingthe severity of a disease; ameliorating or relieving symptoms associatedwith a disease; and causing a regression of the disease or one or moreof the symptoms associated with the disease.

As would be recognized by those of ordinary skill in the art, choleratoxin is an oligomeric protein complex, which is secreted by thebacterium Vibrio cholerae and is thought to be responsible for theenteric symptoms characteristic of a cholera infection. The choleratoxin itself is generally composed of six protein subunits, namely asingle copy of the A subunit, which is thought to be the toxic portionof the molecule responsible for its enzymatic action; and five copies ofthe B subunit, which form a pentameric ring and are thought to comprisethe non-toxic portions of the molecule responsible for binding toreceptors, such as the GM1 ganglioside receptor, which contains aglycosphingolipid (e.g., a ceramide and oligosaccharide) with one sialicacid and which is attached to the surface of a host cell. As such, theterm “cholera toxin B subunit” is used herein to refer to a single Bsubunit of the cholera toxin as well as to B subunits of the choleratoxin in the form of multimers (e.g., in a pentameric form). Exemplarynucleic acid and amino acid sequence of a native cholera toxin B subunitpolypeptide from wild-type Vibrio cholerae are provided herein in SEQ IDNOS: 1 and 2 in the Sequence Listing appended hereto.

The terms “polypeptide,” “protein,” and “peptide,” which are usedinterchangeably herein, refer to a polymer of the 20 protein aminoacids, or amino acid analogs, regardless of its size or function.Although “protein” is often used in reference to relatively largepolypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “polypeptide” as used herein refers to peptides, polypeptides, andproteins, unless otherwise noted. The terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein when referring to a geneproduct. Thus, exemplary polypeptides include gene products, naturallyoccurring or native proteins, homologs, orthologs, paralogs, fragmentsand other equivalents, variants, and analogs of the foregoing. The term“native,” when used with reference to a polypeptide, refers to apolypeptide that is encoded by a gene that is naturally present in thegenome of an untransformed cell.

The terms “polypeptide fragment” or “fragment,” when used in referenceto a reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions canoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both.

A fragment can also be a “functional fragment,” in which case thefragment retains some or all of the activity of the referencepolypeptide as described herein. For example, in some embodiments, afunctional fragment of a cholera toxin B subunit polypeptide can referto a polypeptide in which amino acid residues have been deleted ascompared to the full-length cholera toxin B subunit polypeptide, butwhich retains some or all of the ability of the full-length choleratoxin B subunit polypeptide to bind to a GM1 ganglioside and/or some orall of the ability of the full-length cholera toxin B subunitpolypeptide to attach to a glycan.

The terms “modified amino acid,” “modified polypeptide,” and “variant”are used herein to refer to an amino acid sequence that is differentfrom the reference polypeptide by one or more amino acids, e.g., one ormore amino acid substitutions or additions. A variant of a referencepolypeptide also refers to a variant of a fragment of the referencepolypeptide, for example, a fragment wherein one or more amino acidsubstitutions have been made relative to the reference polypeptide. Avariant can also be a “functional variant,” in which the variant retainssome or all of the activity of the reference protein as describedherein. For example, in some embodiments, the cholera toxin B subunitvariant polypeptides described herein include amino acid sequences inwhich one or more amino acids have been added and/or replaced, but whichnonetheless retain and/or enhance some or all of the ability of thefull-length cholera toxin B subunit polypeptide to bind to a GM1ganglioside and/or some or all of the ability of the full-length choleratoxin B subunit polypeptide to attach to a glycan.

As noted, in some embodiments of the presently-disclosed subject matter,an isolated polypeptide is utilized that comprises a cholera toxin Bsubunit variant polypeptide having one or more modifications. The CTBpmay be produced by any suitable recombinant production platform,including, but not limited to, plants, E. coli, yeast, insect cells,mammalian cells, or a combination thereof. For example, in someembodiments, the one or more modifications increase the expression ofthe polypeptide in a plant cell.

In some embodiments, the one or more modifications to the CTBp include aC-terminal hexapeptide sequence attached to the CTB subunit. In someembodiments, the one or more modifications to the cholera toxin Bsubunit variant polypeptide include an endoplasmic reticulum retentionsignal having the amino acid sequence KDEL (SEQ ID NO: 31). In someembodiments, the KDEL sequence is linked to the cholera toxin by a twoamino acid linker to comprise, in some embodiments, the signal: SEKDEL(SEQ ID NO: 30). In some embodiments, the cholera toxin B subunitvariant polypeptide comprises the amino acid sequence of SEQ ID NO: 4,SEQ ID NO: 6, or SEQ ID NO: 25. In some embodiments, and without wishingto be bound by any particular theory, the addition of an endoplasmicreticulum retention signal, such as the amino acid sequence KDEL or HDELor other similar sequence with similar functions, to a cholera toxin Bsubunit polypeptide enhances the variant polypeptide's wound healing,anti-inflammatory, and anti-cancer activity (e.g., anti-colon canceractivity), as described herein below. In some embodiments, the choleratoxin B subunit variant polypeptide comprises the cholera toxin Bsubunit variant polypeptide describe in, for example, Hamorsky, et al.,“Rapid and Scalable Plant-based Production of a Cholera Toxin B SubunitVariant to Aid in Mass Vaccination against Cholera Outbreaks.” PLoSNTD.March 2013. 7(3): e2046, which is incorporated herein by reference inits entirety.

In some embodiments of the presently-disclosed polypeptides, the one ormore modifications to the cholera toxin B subunit variant polypeptideinclude the addition (e.g., an addition at the N-terminal of the choleratoxin B subunit variant polypeptide) of a secretory signal peptidecapable of transferring or translocating the cholera toxin B subunitpeptide such that the cholera toxin B subunit variant polypeptides isaccumulated in a particular location in a plant tissue, such as in theapoplasts of plant cells. In some embodiments, the secretory signalpeptide is selected from the group consisting of a rice (e.g., Oryzasativa) alpha-amylase secretory signal peptide (e.g., SEQ ID NO: 18), aNicotiana plumbagenifolia calreticulin secretory signal peptide (e.g.,SEQ ID NO: 20), an apple (e.g., Malus domestica) pectinase secretorysignal peptide (e.g., SEQ ID NO: 22), and a barley (Hordeum vulgare)alpha-amylase secretory signal peptide (e.g., SEQ ID NO: 24). In someembodiments, the secretory signal peptide has an amino acid sequenceselected from the group consisting of SEQ ID NOS: 18, 20, 22, and 24. Insome embodiments, the secretory signal peptide comprises a ricealpha-amylase secretory signal peptide, such as the rice alpha-amylasesecretory signal peptide of SEQ ID NO: 18.

In some embodiments, an isolated cholera toxin B subunit variantpolypeptide is utilized that comprises a cholera toxin B subunit variantlinked to a secretory signal peptide, such as those described hereinabove, and an endoplasmic reticulum retention signal. In someembodiments, the variant polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 26-29.

With further regard to the polypeptides of the presently-disclosedsubject matter, in some embodiments, a cholera toxin B subunit variantpolypeptide used in accordance with the presently-disclosed subjectmatter includes one or more mutations so as to include a plurality ofN-linked glycosylation sequons (i.e., Asn-X-Ser or Asn-X-Thr sequences)in the variant polypeptide sequences and thereby provide a mechanism todisplay multiple N-linked H-Man glycans and mimic a virus-likecarbohydrate cluster. In some embodiments, about 1, about 2, about 3,about 4, about, 5, about 6, about 7, about 8, about 9, or about 10N-linked glycosylation sequons are included in an exemplary choleratoxin B subunit variant polypeptide of the presently-disclosed subjectmatter. In some embodiments, a cholera toxin B subunit variantpolypeptide is provided that comprises 2 N-linked glycosylation sequons,such as, in some embodiments, a cholera toxin B subunit variantpolypeptide having the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO:10. In other embodiments, a cholera toxin B subunit variant polypeptideis provided that comprises 3 N-linked glycosylation sequons, such as, insome embodiments, a cholera toxin B subunit variant polypeptide havingthe amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14. In someembodiments, the polypeptide comprises two or more N-linkedglycosylation sequons, such as, in some embodiments, the polypeptides ofSEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

In some embodiments, the presently-disclosed subject matter furtherprovides a number of other benefits or advantages. For example, in someembodiments, and as determined by experimentation, the dosing regimenfor the above-described CTBp-based therapy reduces the dose frequencyand amount compared to current therapeutic options for ulcerativecolitis patients. In some embodiments, the CTBp described herein haswound healing potential not reproducible with native CTB.

For administration of a therapeutic composition as disclosed herein(e.g., a composition comprising a cholera toxin B subunit variantpolypeptide of the presently-disclosed subject matter and apharmaceutically-acceptable vehicle, carrier, or excipient),conventional methods of extrapolating human dosage based on dosesadministered to a murine animal model can be carried out using theconversion factor for converting the mouse dosage to human dosage: DoseHuman per kg=Dose Mouse per kg/12 (Freireich, et al., (1966) CancerChemother Rep. 50:219-244). Drug doses can also be given in milligramsper square meter of body surface area because this method rather thanbody weight achieves a good correlation to certain metabolic andexcretionary functions. Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich, et al. (Freireich etal., (1966) Cancer Chemother Rep. 50:219-244). Briefly, to express amg/kg dose in any given species as the equivalent mg/sq m dose, multiplythe dose by the appropriate km factor. In an adult human, 100 mg/kg isequivalent to 100 mg/kg×37 kg/sq m=3700 mg/m2.

Suitable methods for administering a therapeutic composition inaccordance with the methods of the presently-disclosed subject matterinclude, but are not limited to, systemic administration, parenteraladministration (including intravascular, intramuscular, intraarterialadministration), oral delivery, topical administration, buccal delivery,rectal delivery, vaginal delivery, subcutaneous administration,intraperitoneal administration, inhalation, intratracheal installation,surgical implantation, transdermal delivery, local injection, andhyper-velocity injection/bombardment. Where applicable, continuousinfusion can enhance drug accumulation at a target site (see, e.g., U.S.Pat. No. 6,180,082). In some embodiments, such as those which include apharmaceutical composition comprising a cholera toxin B subunit variantpolypeptide of the presently-disclosed subject matter, thepharmaceutical composition can be administered orally to thereby elicitan immune response.

Regardless of the route of administration, the compounds of thepresently-disclosed subject matter are typically administered in amounteffective to achieve the desired response. As used herein, the terms“effective amount” and “therapeutically effective amount” refer to anamount of the therapeutic composition (e.g., a composition comprising acholera toxin B subunit variant polypeptide of the presently-disclosedsubject matter, and a pharmaceutically-acceptable vehicle, carrier, orexcipient) sufficient to produce a measurable biological response (e.g.,an increase in levels of IgA). Actual dosage levels of activeingredients in a therapeutic composition of the presently-disclosedsubject matter can be varied so as to administer an amount of the activepolypeptide(s) that is effective to achieve the desired therapeuticresponse for a particular subject and/or application. Of course, theeffective amount in any particular case will depend upon a variety offactors including the activity of the therapeutic composition,formulation, the route of administration, combination with other drugsor treatments, severity of the condition being treated, and the physicalcondition and prior medical history of the subject being treated.Preferably, a minimal dose is administered, and the dose is escalated inthe absence of dose-limiting toxicity to a minimally effective amount.Determination and adjustment of a therapeutically effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art.

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902 and 5,234,933; PCT International Publication No. WO93/25521; Berkow, et al., (1997) The Merck Manual of MedicalInformation, Home ed. Merck Research Laboratories, Whitehouse Station,New Jersey; Goodman, et al., (2006) Goodman & Gilman's thePharmacological Basis of Therapeutics, 11th ed. McGraw-Hill HealthProfessions Division, New York; Ebadi. (1998) CRC Desk Reference ofClinical Pharmacology. CRC Press, Boca Raton, Fla.; Katzung, (2007)Basic & Clinical Pharmacology, 10th ed. Lange Medical Books/McGraw-HillMedical Pub. Division, New York; Remington, et al., (1990) Remington'sPharmaceutical Sciences, 18th ed. Mack Pub. Co., Easton, Pa.; Speight,et al., (1997) Avery's Drug Treatment: A Guide to the Properties,Choice, Therapeutic Use and Economic Value of Drugs in DiseaseManagement, 4th ed. Adis International, Auckland/Philadelphia; and Duch,et al., (1998) Toxicol. Lett. 100-101:255-263, each of which areincorporated herein by reference.

As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently-disclosed subject matter. As such, thepresently-disclosed subject matter provides for the treatment of mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like.

The practice of the presently-disclosed subject matter can employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook,Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press,Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I andII, Glover, ed., 1985; Polynucleotide Synthesis, M. J. Gait, ed., 1984;Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells,J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987;Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., AcademicPress Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987; Handbook OfExperimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell,eds., 1986.

The presently-disclosed subject matter is further described by thenon-limiting examples shown below.

EXAMPLES

Materials and Methods for Examples 1-9

A non-glycosylated variant of CTB (CTBp; SEQ ID NOS: 3 and 4) can berapidly and efficiently manufactured in Nicotiana benthamiana plants.That CTBp showed comparable GM1 binding affinity, physicochemicalstability and immunogenicity to native (E. coli produced) CTB.Additionally, antibodies elicited by oral administration of CTBp in micewere able to neutralize the cholera holotoxin. Those results indicatedthat CTBp provides a viable alternative to the recombinant proteinantigen included in Dukoral® oral cholera vaccines, potentiallyfacilitating reactive mass vaccination to respond to cholera outbreaks.Detailed investigation into the biological impacts of orallyadministered CTBp on the GI tract will not only bridge efforts in oralvaccine development, but also fill the aforementioned gap in ourknowledge about one of the most potent mucosal immunomodulatory proteinsknown to date. In this regard, in Examples 1-9, the global impacts ofCTBp oral vaccination on the small intestine and colon werecharacterized by elucidating changes in their immune cell populationsand gene expression profiles as well as the gut microbiome. Cell cultureand animal models were then used to investigate CTBp's oralimmunotherapeutic effects. To this end, a Caco-2 wound healing assay wasused to show enhanced wound healing with CTBp. Additionally, a dextransulfate sodium (DSS) mouse model of intestinal wounding and ulcerativecolitis was used, another major form of IBD to which CTB's impacts havenot been reported before. As described below, the results revealed CTB'spreviously unidentified mucosal wound healing capacity and showedrelevance as an oral immunotherapy for ulcerative colitis andcolitis-associated colon cancer.

Animals. 8 week old C57BL/6J female mice were obtained from JacksonLaboratories (Bar Harbor, Me.) and allowed to acclimate for one week.

Induction of colitis and study design. For the acute DSS studies,animals were vaccinated with PBS or 30 μg plant-made cholera toxin Bsubunit (CTBp) two times prior to DSS exposure. Vaccinations occurredtwo weeks prior to and at the initiation of DSS exposure. The DSSexposure was slightly modified based on a previously published protocol.Animals were exposed to 4% DSS in drinking water for up to eight daysand allowed up to a seven day recovery period in which the animalsreceived normal drinking water. Mice were sacrificed using carbondioxide inhalation followed by a thoracotomy. Serum, Fecal and Colonsamples were taken for further analysis.

For the colon cancer study, Azoxymethane (AOM; 10 mg/kg) wasadministered by intraperitoneal injection one week after arrival. DSSexposure (2%) was initiated one week after the AOM injection for sevendays and allowed to recover for 14 days following the DSS exposureperiod. The DSS exposure and recovery cycle was repeated three times andmice were sacrificed by Carbon Dioxide inhalation followed bythoracotomy following the 3^(rd) cycle. This method was slightlymodified from the published protocol. Colons were excised for furtheranalysis. Animals were housed according to the University ofLouisville's Institutional Animal Care and Use Committee standards.

RNA isolation. Sections from the Small Intestine and Distal colon werestored in RNAlater™ (Qiagen, Valencia, Calif.) at −20° C. until RNA wasisolated. Colon Tissue (approximately 14 mg) was placed in QIAzol lysisreagent in a 2.0 mL conical bottom centrifuge tube with Zirconia/Silicabeads. A Bead Beater was used to homogenize the tissue. An RNeasyMicroarray Tissue Kit from Qiagen (Catalog no. 73304) was used to purifythe RNA from the tissue homogenate. RNA was stored at −80° C. untilused.

Microarray gene expression analysis. Total RNA was amplified and labeledfollowing the Affymetrix (Santa Clara, Calif.) standard protocol forwhole transcript expression analysis, followed by hybridization toAffymetrix Mouse Gene 2.0 ST® arrays. The arrays were processedfollowing the manufacturer recommended wash and stain protocol on anAffymetrix FS-450 fluidics station and scanned on an AffymetrixGeneChip® 7G scanner using Command Console 3.3. The resulting .cel fileswere imported into Partek Genomics Suite 6.6 and transcripts werenormalized on a gene level using RNA as normalization and backgroundcorrection method. Contrasts in a 1-way ANOVA were set up to compare thetreatments of interest.

Quantitative RT-PCR Gene expression analysis. Gene expression wascarried out by quantitative RT-PCR using quality verified RNA samplesobtained previously. First strand cDNA was obtained from reversetranscription of 150 ng RNA using a SUPERSCRIPT VILO cDNA synthesis kit(Life Technologies) according to the manufacturer's instructions.Optimal amounts of template cDNA were added to a reaction mixturecontaining 10 μl of 2× TaqMan® Fast Advanced Master Mix (LifeTechnologies) and endonuclease free water to 20 μl and loaded in TaqMan®Array Standard 96 well Plates (Applied Biosystems). Those platescontained pre-spotted individual TaqMan® Gene Expression probes fordetection of genes of interest as well as the house keeping genes 18 S,beta actin (ACTB), and GAPDH. PCR amplification was carried out using a7900HT Fast Real-Time PCR System (Applied Biosystems) in the followingconditions: 95° C., 20 min; 40 cycles (95° C., 1 min); 20 min at 60° C.The 7500 Software v2.0.6 (Applied Biosystems) was used to determine thecycle threshold (Ct) for each reaction and derive the expression Ratios.

Lymphocyte isolation. Eight week old C57BL/6J female mice werevaccinated according to the schedule in the Animals and Treatmentssection. One or two weeks following the second vaccination with PBS orCTBp the animals were sacrificed. Colons were removed for immune cellisolations. Lamina propria lymphocytes (LPLs) were isolated from thecolons by using a series of washing steps and collagenase steps.Epithelial cells, mucus and fat tissue were removed by incubating withEDTA at 37° C. The colon and small intestine were cut into small piecesand incubated with collagenase at 37° C. Cell suspensions were separatedusing Percoll gradients and the middle layers, containing thelymphocytes, were isolated and counted. Cells were counted in ahemocytometer.

Flow Cytometry. Cells were stained using antibodies and a Cell stainingkit from eBiosciences, Inc. (San Diego, Calif.). Briefly, tubescontaining 1×10⁶ cells were washed with flow cytometry staining buffer 2times. Fc Block was added to each tube in flow cytometry staining bufferfor 10 minutes.

For adaptive immune cell populations, surface staining antibodies werethen added to each tube (CD3-FITC, CD4-APC-Cy7, CD25-PerCP) and allowedto incubate at 4° C. for 30 minutes. After removing surface antibodies,fixation/permeabilization buffer was added to the tubes and incubatedovernight. The following morning the tubes were washed withpermeabilization buffer two times and again incubated for 10 minuteswith Fc block. Internal cell antibodies (Gata3-PE, T-Bet-PE-Cy7,FoxP3-APC, IL-17-eFlour450) were added to each tube and incubated for 30minutes at 4° C. The tubes were washed two times with permeabilizationbuffer and finally cells were suspended in flow cytometry stainingbuffer.

For innate immune cell populations, surface staining antibodies wereadded to each tube (CD19-APC, CD3-FITC, CD49b-PE, F4/80-PeCy7,CD11c-PerCP-Cy5.5, CD8-APC-eFluor 780, and CD45-eFlour450) and allowedto incubate at 4° C. for 30 minutes. After removing surface antibodies,fixation buffer was added to the tubes and incubated overnight. Thetubes were washed two times with flow cytometry staining buffer andsuspended in flow cytometry staining buffer. Events (1×10⁵) were countedon a BD FACSCanto™ II and analyzed with the BD FACSDiva Software v6.1.3.

Caco-2 Wound Healing Assay. The Caco-2 wound healing assay was performedusing a modified method. Briefly, Caco-2 cells were seeded and grown toconfluence in 6 well plates (Thermo Scientific™ Nunc™ Cell-CultureTreated). The culture medium was discarded and 0.5-1.0 mm across linearwounds were made with a 200 μL sterile beveled pipette tip (USAScientific) and cells were washed with PBS to remove loose cells. CTBp(0.3-1 μM), transforming growth factor-β (TGF-β; 5 ng/ml), or PBS weresubsequently added in fresh serum-deprived medium to a total volume of 2mL. Photomicrographs of the wounds were taken at 0 hours and 48 hoursafter the wounding using a 4× magnification. Quantification of theremaining cell-free area to the initial wound area was calculated as amean percentage using the public domain software Image J.

Histology. Colons were removed and washed with PBS. A portion of thedistal colon was fixed with paraformaldehyde overnight and stored in 70%ethanol until paraffin embedding, sectioning and routine H&E staining.Inflammation scoring was performed using a scale that has beenpreviously published. Tissue sections from 8 mice were scored andaveraged for each group. Statistics were performed comparing each group.

Protein Isolation and quantification. Distal colon sections wereisolated at sacrifice and stored at −80° C. until the protein wasisolated and analyzed. Briefly, tissue was frozen in liquid nitrogen andpulverized with a Bessman Tissue Pulverizer and placed in T-PER (ThermoScientific) with a protease inhibitor cocktail (Sigma-Aldrich). Proteinwas isolated by gravity centrifugation of tissue fragments, removing thebuffer containing isolated protein, and storing at −80° C. untilanalysis. Protein sample concentrations were determined using a Nanodrop1000 (Thermo Scientific). Protein was normalized for all samples priorto loading on a Mouse Cytokine/Chemokine Magnetic Bead Panel (EMDMillipore). The panel was analyzed with a Milliplex MAP Kit on a MagPixwith Luminex xMAP technology.

Disease Activity Index. Animals were scored on a daily basis whichconsisted of the following scoring rubric adapted from the literature.Weight loss: 0 for no weight loss, 1 for 1 to 5% weight loss, 2 for 6 to10% weight loss, 3 for 11 to 15% weight loss and 4 for greater than 15%weight loss. Stool consistency: 0 for normal stools, 2 for loose stoolsand 4 for diarrhea. Occult blood: 0 for no blood, 1 for some occultblood, 2 for heavy positive occult blood, 3 for visible blood in stoolwith no anus clotting, or 4 for gross anus bleeding and clottingpresent.

Tumor Scoring. Tumors were scored via endoscopic analysis of the fulllength of the colon. Tumor scoring was based on the following rubric: 0for no tumor, 1 is a very small but detectable tumor, 2 the tumor coversup to ⅛ colon circumference, 3 tumor covers ¼ of colon circumference, 4tumor covers up to ½ of colon, and 5 tumor covers more than ½ of colon.

Statistics. Graphs were prepared and analyzed using Graphpad Prismversion 5.0 (Graphpad Software). To compare two data sets, an unpaired,two-tailed Student's t test was conducted. To compare three or more datasets, a one-way ANOVA with a Bonferroni post-test was conducted. Forbody weight and DAI results, a Two-way ANOVA with a Bonferroni post-testwas conducted.

Example 1—Colon Lymphocyte Profile was Significantly Altered by CTBp

Since CTB is a strong mucosal immunogen and induces a robust mucosalantibody response upon oral administration, the immune cell populationswere characterized in various immune compartments in CTBp-vaccinatedmice. Both innate and adaptive immune cell populations were evaluated inthe small intestine lamina propria and colon lamina propria, mesentericlymph node, Peyer's patches and spleen using flow cytometry. For thisanalysis, mice were given PBS or 30 μg CTBp, orally, twice over atwo-week period, a standard regimen used for oral cholera vaccination,and sacrificed one or two weeks after the second dose. Small intestinesand colons were excised and Peyer's patches were removed from the smallintestines. Leukocytes were isolated from the small intestine and colonlamina propria, Peyer's patches, mesenteric lymph node and spleen, andwere then stained with specific markers for adaptive and innate immunecell subsets.

T cell profiles were not significantly affected in the mesenteric lymphnodes or the spleen (FIGS. 1 and 2). A temporary significant increase ofthe B cell population (CD19+) within CD45+ cells was noted in Peyer'spatches one-week post CTBp administration. For effector sites (laminapropria), an increased proportion of FoxP3+ CD25+ regulatory T (Treg)cells among total CD3+CD4+ T cells was noted in the small intestine oneweek post vaccination, and there was also a trend of increase in CD8+ Tcells (P=0.0667; FIG. 1). Meanwhile, a significantly increasedproportion of T_(H)1 (TBet+) cells within CD3+CD4+ cells was noted inthe colon lamina propria one week post-CTBp administration (FIG. 1). Inboth small intestine and colon lamina propria, however, the changes in Tcell populations did not sustain through the next week (FIG. 2),suggesting that CTBp oral administration has relatively short-termimpacts on T cell profiles while inducing a robust and durable antibodyresponse.

Unexpectedly, more significant impacts were noted in innate immune cellpopulations in the lamina propria of colon, but not of small intestine,two week post CTBp oral administration (FIG. 3); macrophages (F4/80+),dendritic cells (CD11c+) and natural killer cells (CD49b+) weresignificantly increased within CD45+ cell populations when compared tothe PBS group, which was associated with significant decrease in Bcells. Immunohistochemistry analysis also revealed that the number ofmacrophages was increased in the colon lamina propria at the same timepoint after CTBp oral administration, compared to the PBS-fed control(FIG. 3). Hence, the results revealed an interesting effect of orallyadministered CTBp on the distal part of the GI tract two weeks afterCTBp administration.

Example 2—Oral CTBp Administration Affects Colon Gene Expression Profilemore than Small Intestine's

A microarray analysis of transcripts isolated from the small intestineand colon was performed to determine if CTBp affected gene expression inthe GI tract, using a Affymetrix Mouse Gene 2.0 ST® array and PartekGenomics Suite 6.6. A heat map was generated (FIG. 4A) to compare thegene expression profiles in the colon and small intestine. Mice wereagain given PBS or 30 μg CTBp twice over a two-week period andsacrificed two weeks after the second dose to further evaluate the timepoint of greatest change in immune cell populations.

Orally administered CTBp had profound impacts on the gene expression ofthe entire intestinal epithelia (FIG. 4B). Interestingly, the geneexpression profile in the small intestine clustered more closelytogether than that of the colon based on the heat map analysis. The geneexpression patterns of the colons from PBS-fed mice were closelyassociated with those of the small intestine from both groups. However,the gene expression in colons from the CTB-treated mice was completelyseparated from the other samples in the heat map analysis (FIG. 4A). Ata global level, 871 genes were significantly (p≤0.01) altered followingCTBp vaccination in the colon, while 184 genes were significantlyaltered in the small intestine (FIG. 4B). Of these significant genes,539 were induced and 332 were suppressed in the colon. By comparison,the small intestine was fairly evenly split between induced andsuppressed genes, 97 and 87, respectively.

Example 3—CTBp Enhances TGFβ-Associated Gene Expression Pathways in theColon

Pathway analysis software in MetaCore™ (version 6.22 build 67265) wasused to dissect the gene expression alterations in the colon by CTBpadministration. Interestingly, TGFβ-dependent pathways heavily populatedthe most significantly induced pathways following CTBp oraladministration (FIG. 4C). Extracellular matrix remodeling pathways andepithelial to mesenchymal pathways were among the most significantlyinduced pathways by CTBp vaccination. Indeed, when evaluating individualgene expression from the microarray analysis Tgfβ1, TgfβII receptor, andSmad4 are significantly induced by CTBp oral administration (FIG. 5).These results indicated that CTBp can facilitate epithelial woundhealing.

By contrast, such strong induction of TGFβ-related pathways was notobserved in the small intestine. Suppressed pathways in the colonepithelia included several metabolic pathways, cystic fibrosistransmembrane conductance regulator (CFTR) pathways, and an apoptosisassociated pathway. Genes associated with lipid, bile acid, pyruvate,and androstenedione and testosterone metabolic pathways weresignificantly blunted by CTBp. Interestingly, Hsp70, Hsp90, Hsp90a, andSt/1 were significantly suppressed in several of the pathways analyzedfollowing CTBp oral administration, which are implicated in theprogression of colon cancer. To confirm the results in our microarrayanalysis quantitative real-time-PCR (qPCR) analysis was performed on thetranscripts of selected induced, suppressed, or unchanged genes. Highagreement was found between the microarray and qPCR results (FIG. 6).Notably, a wound healing pathway-focused qPCR analysis revealed thatmany of the key genes in this pathway, including: Col14a, Mmp2, Col1a1,and Col3a1 are significantly upregulated by CTBp oral administration(FIG. 6). Based on the changes in immune cell populations and geneexpression it was hypothesized that the gut microbiome may also bealtered.

Example 4—The Overall Microbiome Profile was Largely Unaltered by CTBp 2Weeks after Administration

With the changes in the gene expression of the colon and immune cellpopulations, a possible change in the microbiome profile was speculated.Fecal samples were collected prior to initial dosing with PBS or CTBpand at the time of sacrifice and sequencing of the V4 region of 16Sribosomal RNA was performed on fecal DNA.

The overall microbiome profile was not significantly altered by CTBporal administration after 2 weeks (FIG. 7A). Bacteroidetes andFirmicutes spp. dominated the abundance levels at the phylum level whichis typical for C57BL/6J mice. Additionally, the six most common specieswere split evenly between Bacteroidetes and Firmicutes (FIG. 7B).However, there were minor subpopulations of gut flora that appeared tohave been modified by CTBp administration; all belonging to the phylumFirmicutes. Of the 12 Operational Taxonomic Units (OTUs) significantlyaltered by CTBp administration, 11 were significantly downregulatedcompared to the PBS colon fecal samples (FIG. 7C). All 11 downregulatedOTUs can be traced to the order Clostridiales of the Firmicutes phylum.The remaining OTU, which was induced, belonged to the orderErysipelotrichales of the Firmicutes phylum.

Example 5—Human Colonic Epithelial Ccell Wound Healing was SignificantlyEnhanced by CTBp

To investigate the mucosal wound healing potential of CTBp suggested bythe gene expression analysis, the human colon epithelial cell lineCaco-2 wound healing model was employed. In this assay, closure of woundafter 24 hours in the presence of CTBp, TGFβ or PBS control wasmeasured.

As shown in FIG. 8, CTBp (1.0 and 3.0 μM) significantly enhanced woundhealing as compared to the PBS control, which was comparable to 5 ng/mlof TGFβ. Co-incubation of CTBp with an anti-TGFβ antibody (50 ng/mL)completely blunted the wound healing, demonstrating that CTBp's effectis mediated by TGFβ.

Example 6—CTBp Mitigated DSS-Induced Acute Colonic Injury andInflammation

With the above data supporting a mucosal wound healing potential ofCTBp, from both colon gene expression analysis after CTBp oraladministration and an in vitro human colon epithelial wound model, itwas next explored if this potential can be translated into a therapeuticeffect in vivo. The DSS model of colitis induces injury in the distalcolons of mice. PBS or 30 CTBp was orally administered twice to miceprior to DSS exposure and body weights were recorded daily. Distalcolons were removed, paraffin embedded and stained with hematoxylin andeosin (H&E) for inflammation scoring.

DSS exposure resulted in significant body weight loss, a strongindicator of disease severity, when compared to control animal's at themost extreme point of weight loss (FIG. 9A). However, oraladministrations of CTBp significantly blunted the weight loss during theend of the DSS exposure and early recovery period.

Histopathological examination was performed on the H&E-stained colontissue sections to assess disease severity at the end of the DSS cycleand after a one-week recovery period. PBS administration resulted inaberrant loss of crypts with crypt regeneration, mild inflammatoryinfiltrates noted, and ulceration present in the colon sections after a1 week recovery period. Oral administration of CTBp resulted inshortening of basal crypts and mild inflammatory infiltrates, but noloss of the epithelial surface following a one week recovery period.(FIGS. 9B and 9C). Importantly, CTBp administration appeared to haveprevented the formation of fibrosis in the submucosa according toMasson's trichrome stain; on the contrary, fibrosis was evident in thePBS-administered control group (FIG. 9C).

Given that CTBp administration significantly improved the recovery fromDSS-induced acute injury and inflammation in the colon, the effect atthe maximum injury/inflammatory point immediately after DSS exposure wasinvestigated next. Indeed, at this point the inflammation score wasindicative of almost complete absence of crypts, moderate inflammatorycell infiltration in the mucosa and submucosa, and loss of theepithelial surface cells with PBS administration (FIG. 10A). Notably,CTBp administration significantly blunted the inflammation score;characterized by a shortening of the basal crypts, mild inflammatoryinfiltrates in the mucosa and submucosa, but retention of the epithelialcell surface. Meanwhile, daily oral administration of 100 μg mesalamine(MES) for 7 days during the DSS exposure, which simulates a currenttreatment for UC in humans, showed similar protection observed with theCTBp administration regimen employed here, demonstrating theeffectiveness of CTBp against acute colonic injury and inflammation.

To further characterize CTBp's protective effect at the gene expressionlevel, changes in ulcerative colitis-related genes were analyzed byquantitative real-time PCR analysis on total RNA isolates from the mousecolons. While the body weight has been mostly recovered at one weekafter the end of DSS exposure, classic ulcerative colitis cytokines,including Il-1β, Tnfα, and Il-33, were still significantly high at thistime point in the PBS-administered group (FIG. 9D). Notably, CTBpadministration blunted the expression of these genes to near baselineone week after the end of the DSS exposure (FIG. 9D). Nlrp3, crucial togut homeostasis, gene expression was significantly induced by DSSexposure and CTBp administration significantly blunted the geneexpression compared to PBS pretreated, DSS exposed mice after a one weekrecovery period. Gm-csf, which induces a proinflammatory cytokineprofile from M1 macrophages, was significantly elevated by DSS exposureand CTBp administration significantly blunted Gm-csf expression.

Overall similar trends were also noted at the maximal inflammatory timepoint; immediately following the end of DSS exposure, CTBpadministration significantly blunted the increase of Il-1β, Il-33, Ifnγ,and Tnfα gene expression induced by DSS exposure (FIG. 10B). Though notstatistically significant, CTBp also alleviated the massive increase ofIl-6 induced by DSS exposure. Additionally, CTBp administrationprevented the elevation of Gm-csf following DSS exposure (FIG. 10B).

To confirm the gene expression results, soluble inflammatory markerswere measured at the protein level in the distal colon using a MilliplexMAP Kit. Immediately following the end of the DSS exposure period, CTBpadministration mitigated increases in major inflammatory proteins(IL-1β, IFNγ, IL-3, IL-4 and IL-5) and IL-3 following DSS exposure aswell as a significant reduction in IL-3 (FIG. 10C). Interestingly, CTBpadministration did not induce IL-10, which was significantly increasedin the DSS exposed PBS pretreated group, suggesting that CTBp'santi-inflammatory activity is independent of the anti-inflammatorycytokine. By the end of the one-week recovery, all the elevatedinflammatory markers induced by DSS exposure had returned to normallevels comparable to those of the non DSS-exposed control group. Hence,there was no noticeable effect of CTBp at the protein level at this timepoint (FIG. 9E).

Example 9—CTBp Oral Administration Protected Against Colon CancerDevelopment in an AOM/DSS Colitis Model

The significant protection seen in the acute colitis/colon injury modelprompted us to investigate if CTBp could also protect in a chronic modelof ulcerative colitis and colitis-associated colon cancer. AOM wasadministered by intraperitoneal injection and one week later mice wereexposed to DSS for one week. The DSS exposure was followed by a two-weekrecovery phase, and the DSS exposure/recovery cycle was repeated anadditional two times. Upon completion of the third cycle, the mice weresacrificed and colons removed for analysis. Disease activity index wasused as a metric to assess colitic activity in mice during the livephase of the experiment, in which occult blood, body weight, and fecalconsistency were scored and averaged for each animal. At sacrifice,colons were analyzed for tumors with an endoscope and tumors were scoredbased on a previously developed rubric.

As shown in FIGS. 11A-11D, DSS exposure significantly and progressivelyincreased the disease activity index score in mice over the 3 exposureperiods in the chronic colitis model. Additionally, tumor numbers weresignificantly increased in mice exposed to DSS compared to controls.Notably, CTBp administration significantly decreased the diseaseactivity index score immediately following the first dose of CTBp andmore dramatically during the 3^(rd) DSS exposure period (FIG. 11A). Thisdecrease in disease activity index score was associated with asignificantly reduced number and size of tumors in CTBp treated micecompared to PBS treated mice. DSS exposed mice developed on average fourtumors per colon over the length of the study (FIG. 11C). CTBpadministration significantly blunted the number of tumors toapproximately one tumor per colon (FIG. 11C). Additionally, asignificant increase of grade 2 and 3 tumors in DSS exposed mice was notseen in mice given CTBp. Of note, grade 4 and 5 tumors were found in DSSexposed mice, but no tumors above a grade 3 were found following CTBpadministration (FIGS. 11C and 11D).

Discussion of Examples 1 -9

The generation of CTBp, a novel CTB variant that was robustly producedin N. benthamiana while retaining key molecular properties of theoriginal protein, has been previously reported. The plant-producedprotein had Asn4→Ser mutation to avoid N-glycosylation and a C-terminalhexapeptide sequence attached for endoplasmic reticulum (ER) retentionin plants to facilitate recombinant production. Notwithstanding thosesequence modifications, CTBp had the GM1-ganglioside binding affinityand capacity to induce mucosal and systemic anti-CTB antibodies thatwere comparable to those of bacterially produced CTB, suggesting thatthey are immunologically identical. CTB's strong mucosal immunogenicityis appreciated. However, its detailed biological activities and impactson the GI tract upon oral administration are not comprehensivelydescribed. Hence, the initial goal for this Example was to characterizewhat affect CTBp had on the GI tract following oral administration usinga systems biology approach; by profiling gene expression changes andcharacterizing lymphocyte populations in relevant immune compartments.The Example also linked gene expression and immune cell populationchanges to the gut microbiota. To effectively reveal CTBp's impacts,mice were administered 30 μg CTBp twice over a two week period, sincethis dose induced the maximum antibody response that lasted over 6months.

It has been previously suggested that CTB alters the T cell profile. Inthe present studies, some significant changes were observed in T_(H)cell compositions in the lamina propria effector sites of the smallintestine and the colon one week after the second oral dose of CTBp(i.e., increase of FoxP3+CD25+ Treg cells in the small intestine andT_(H)1 cells in the colon; FIG. 1). However, these changes in T_(H) cellpopulations did not persist through the second week post CTBp oraladministration despite a long-lasting intestinal and systemic anti-CTBantibody response induced by the same dosing regimen.

The most striking changes were noticed in the colon lamina propria 2weeks after CTBp administration; interestingly, while T cell profileswere not affected, other immune cell types were significantly altered inthe colon (FIG. 1). Dendritic cell, macrophage and natural killer cellratios among the CD45+ cell population were significantly increased byCTBp administration. On the contrary, B cell composition wassignificantly reduced, although this may simply reflect the relativeincrease of the innate immune cell populations among CD45+ cells (Bcells still represented the major fraction (>50%) of CD45+ cells). Sucha finding on orally administered CTB's impacts on innate immune cells inthe distal portion of the GI tract mucosa is unprecedented, representingone of the protein's yet to be uncovered immunomodulatory activities.

In vitro, it is appreciated that CTB affects dendritic cell maturationand diminishes the proinflammatory response of macrophages tolipopolysaccharide. However, whether these in vitro results are relevantto the present findings in vivo is elusive, and additional investigationis warranted for the detailed mechanism and the biological consequenceof CTBp's impacts on innate immune cells in the colon. The results ofthis Example, however, showed that the alteration in immune cellpopulations in the colon was associated with dramatic changes in thegene expression pattern of the colonic epithelium (FIG. 4), but it didnot seem to be linked to gut microbiota (FIG. 7).

The transcriptomic analysis revealed that CTBp oral administrationdramatically altered gene expression profiles in the small intestine andcolon of mice. However, of interest is the findings that the greatestchange was noted in the colon and that genes affected by CTBp arecompartmentalized between the two intestinal regions (FIG. 4). As geneexpression changes in the colon were deciphered, it was found thatTgfβ-mediated pathways were most significantly altered in the colon, butthis was not observed in the small intestine. Additionally, a pathwayfocused qPCR analysis revealed significant upregulation of genesassociated with wound healing (FIG. 4C).

In a model of colonic anastomosis in rabbits, CT was previously shown toenhance wound healing by inducing Tgfβ. The results of this Examplesuggest that the effect could have been mediated in part by the Bsubunit of CT in that model. Although recombinant CTB was previouslyshown to induce IgA class-switching through Tgβ1 stimulation, theforegoing studies were believed to be the first report showing that CTBporal administration increased Tgβ1 expression in the colon epithelium.TGFβ is a pleiotropic cytokine involved in various biologicalactivities, including wound healing pathways. The present data combinedwith these previous findings point to an anti-inflammatory potential aswell as a major role for CTBp in altering TGFβ-driven pathways in thecolon. Conversely, the results of this Example revealed CTBp's novelfunction to enhance wound healing, apart from eliciting a strong humoralimmune response, via its capacity to induce Tgfβ expression.

Despite these changes to gene expression and innate immune cellpopulations, no overall change in the microbiome of mice treated withCTBp (FIG. 7) was observed. However, there were changes at the specieslevel in 12 OTU's with 11 being of the Clostridiales order. Thissuggests that CTBp may have a transient effect on specific gutmicrobiome subpopulations but overall, the familial populations are notaffected, at least at the specific time point (two weeks post CTBp oraladministration) analyzed in the present study. While a potential longterm effect on the microflora is yet unknown, the results of thisExample indicate that the drastic changes in the colon immune cell andgene expression profiles observed two weeks post CTBp oraladministration resulted from the protein's direct impacts on the colonicmucosa.

Consistent with the gene expression pathway analysis of the microarrayand qPCR results, an in vitro wound healing assay using the Caco-2 humancolon epithelial cell line showed that CTBp could enhance TGFβ-mediatedmucosal wound healing (FIG. 8). This unique wound healing enhancementcapacity of CTBp was further demonstrated in a DSS-mediated acutecolitis model in mice. Hallmarks of decreased inflammation (decreasedbody weight loss and inflammation scoring) in the DSS colitis model werenoted after CTBp administration (FIG. 9). At the protein level, IL-3IL-5, and GM-CSF are significantly elevated at the end of the DSSexposure period in PBS pretreated mice (FIG. 10), which is indicative ofeosinophil recruitment to the distal colon and increased inflammation.However, these proteins were not significantly increased in CTBp treatedmice immediately following the end of the DSS exposure; suggesting CTBpis able to blunt inflammation in colitic mice. Elevated GM-CSF has alsobeen shown to lead to enhanced tumorigenisis in the colon.

Notably, after one-week recovery from DSS exposure, genes associatedwith fibrosis were significantly blunted by CTBp administration (FIG.9). Tgfβ1, Il-1β, and Nlrp3 play significant roles in promoting collagendeposition leading to fibrosis, which results from increased expressionof collagen 1 (Col1a1), the major fibrous collagen (32, 42). Also, Nlrp3suppression has previously been shown to blunt injury in the DSS colitismodel of ulcerative colitis. In fact, histological analysis demonstratedthat CTBp administration facilitated recovery of the colonic epithelialdamage (FIG. 9) and prevented fibrosis induced by DSS exposure. Itshould be noted that the TGFβ- and collagen-inducing effects of CTBpobserved under normal physiological conditions did not result in adverseeffects in the acute colitis model, perhaps due to the dose used in thestudy was within a therapeutic range in which the protein did notoverstimulate the activation of TGFβ pathways. In turn, these resultsrevealed the therapeutic effects of CTBp for mucosal wound healing andcolitis.

The DSS colitis model employed in the present study was awell-established method for the study of ulcerative colitis and woundhealing in a mouse. IBD is composed of two main disease subcategories,Crohn's disease and ulcerative colitis, which have similaritiespathologically but some important differences remain. Overall,differences exist in genetic susceptibility, environmental factors andlastly different T helper cell expression profiles. While the exactetiology of IBD remains elusive, Crohn's disease is generally acceptedto be a T_(H)1 driven disease while ulcerative colitis is a T_(H)2driven disease with T_(H)17 cells appearing to have a role in both IBDsubtypes. More recently, others have found that a subset of Th17 cellsthat coproduce IFNγ and IL-17 were specifically enriched in both activeCrohn's disease and ulcerative colitis; highlighting the uncertaintyremaining in IBD pathogenesis.

Ulcerative colitis has become a growing concern in the developed worldand colon cancer is the second leading cause of cancer-related death inthe United States. Additionally, individuals with ulcerative colitis areat an enhanced risk for developing colon cancer. Extent and duration ofthe inflammation in the colon plays a clear role in the development ofcolorectal cancer, which suggests that, by decreasing the length of theinflammation period and severity of the inflammation, the incidence ofcolorectal cancer can be decreased. The findings that CTBp mitigatedinflammation and enhanced wound healing in an acute DSS model suggestedthat the protein could also protect in a more chronic colitis stateleading to colorectal cancer development.

In the chronic colitis/colon cancer model, disease activity indexscoring revealed significant protection by CTBp immediately followingdosing and during the third cycle of DSS exposure, suggesting that theprotein's therapeutic effect took place relatively quickly afteradministration. This in turn provided an implication for optimal CTBpdosing schedule for potential immunotherapy against ulcerative colitis.Data indicated that CTBp most effectively induced anti-colitis activitywhen dosed at the time of ongoing, not prior to the onset of,inflammation. Most importantly, biweekly oral administration of CTBpduring the induction of chronic colitis resulted in significant decreasein the tumor number and tumor score at the end of the study. Thesefindings illustrate that CTBp's effect on the wound healing pathway notonly protects against inflammation and enhances wound healing but alsoprotects against the development of colon cancer.

Interestingly, the microarray analysis of colon gene expression inhealthy mice (FIG. 4) revealed that Hsp70 and Hsp90 were significantlysuppressed by CTBp administration; increased expression of these stressproteins has been associated with poor outcomes in colon cancer.Consequently, there may be an additional mechanism for CTBp to slow thegrowth of tumors in colorectal cancer besides anti-inflammatory/woundhealing effects against colitis. No major difference was observedbetween the two doses of CTBp (i.e., 3 and 10 μg per mouse) in tumorprevention activity. In fact, the disease activity index was slightlybetter (though not statistically significant) with the lower dose. Theapparent lack of dose-dependent effect may be related to the therapeuticwindow of CTBp, although a more thorough study is necessary to derive aconclusion in this regard.

Recently, the whole CT molecule (not CTB) has been shown to protectagainst colon carcinogenesis in the DSS colitis model employing Balb/cmice. In that study, 10 μg of CT was orally administered at the firstday of each of the three cycles of DSS exposure. Interestingly, theauthors found that macrophages were upregulated by CT administrationmuch like we found with CTBp administration. However, the authorsobserved upregulated regulatory T-cells and IL-10 in the colonic mucosa,while such an effect was not seen with CTBp. This suggests the distinctmechanisms of CT and CTB in colitis-associated colon cancer prevention.Since CTBp is not toxic, unlike CT, and the effective dose of CTBp seemsto be comparable to that of CT (total 4 administrations of 3 and 10 μgCTBp vs. total 3 administrations of 10 μg CT), it appears that CTBp maybe superior as a candidate drug against ulcerative colitis andcolitis-associated colon cancer.

Taken together, these results show that oral administration of CTBp hasprofound impacts on the distal portion of the GI tract in innate immunecell and epithelial gene expression profiles, which led us to discoverCTBp's unique potential as a novel mucosal wound healing agent. Coupledwith the efficient recombinant production in plants, CTBp represents anorally active immunotherapeutic agent providing clinical benefits forpatients with ulcerative colitis.

Example 10—Cholera Toxin B Subunit Protects against Colitis-AssociatedColon Cancer in a Mouse Model

Inflammatory Bowel Disease (IBD) is a growing problem in the developedworld and a significant risk factor for developing colorectal cancer(CRC). Cholera holotoxin (CT) is the causative agent of severe diarrheaupon infection with Vibrio cholerae. CT induces massive immune responsesat exposed mucosa and is one of the most potent mucosal immunogensdescribed to date. A well characterized model of Ulcerative Colitis (UC)in mice is the DSS model, which induces a similar response as human UC(T helper 2 mediated). In a chronic DSS model in BALB/cJ mice, CT hasalso been shown to suppress carcinogenesis. Additionally, the B Subunitof CT (CTB) is currently used as a component of an internationallylicensed, World Health Organization (WHO)-prequalified oral choleravaccine for human use (Dukoral®, Crucell). In a Crohn's Disease mousemodel, CTB has been shown to protect against inflammation. This work hasled to a clinical trial in which CTB was able to blunt inflammation inhumans as well. The instant inventors have generated, in plants(Nicotiana benthamiana), a robust recombinant production system for anon-glycosylated variant of CTB (CTBp). The potential for massproduction of CTBp led the inventors to explore the anti-inflammatoryand immunosuppressive activities demonstrated by original CTB. In thisExample, whether CTBp could protect against colitis-associated coloncancer in a mouse model was investigated.

Animals and treatments. Eight week old female C57BL/6J mice wereobtained from Jackson Laboratory (Bar Harbor, Me.). For the DSSexperiment, animals were injected intraperitoneally (i.p.) withAzoxymethane (AOM) (10 mg/kg) one week prior to a one-week 2% dextransulfate sodium (DSS) exposure period. The animals were given two weeksto recover from DSS exposure and the three-week cycle was repeated twomore times. At the end of the first DSS exposure period mice were givenoral (gavage) doses of CTBp (3 or 10 μg) or PBS and continued biweeklyuntil the end of the study (FIG. 12). Body weights and Disease ActivityIndex (DAI) were determined daily. Following the last recovery period,the mice were sacrificed by CO2 asphyxiation, the colons were excised,and the tumors were scored. Colon tissue was analyzed for inflammation,gene expression and inflammatory cytokine levels.

Hematoxylin and Eosin Staining. Tissue sections were collected from thedistal colon and placed in 10% formalin for 18 H. The tissue was thenplaced in 70% Ethanol until the time of paraffin embedding. Paraffinembedding, cutting and H&E staining were performed by a trainedprofessional. Tissue sections were scanned on an Aperio Scan Scope CSfor analysis. Tissue sections were scored based on a modifiedprestablished rubric.

Disease Activity Index Scoring. Body weights and fecal samples werecollected daily from the start of the first DSS exposure until studytermination. The scoring rubric included a score for body weight loss: 0for 0%, 1 for 1 to 5%, 2 for 5 to 10%, 3 for 10 to 15% and 4 for >15%weight loss. Additionally, fecal consistency was scored as: 0 for normalstool, 2 for loose stool and 4 for diarrhea. Finally occult blood wasscored as: 0 for no blood, 1 for positive test, 2 for maximum positivetest, 3 for visible blood in feces, 4 for gross anus bleeding andclotting present. The scores were added and averaged for each mouse andplotted.

Tumor Counting. At sacrifice, the colons were removed and an endoscopewas inserted in the rectum and the entire length of the colon wasscanned. Following the sacrifice the video was reviewed and tumors werescored based on the following rubric: 0=no tumor, 1=small butdetectable, 2=covers up to ⅛ of colon, 3=covers up to ¼ of colon,4=covers up to ½ of colon, and 5=covers over ½ of colon circumference.

Gene Expression. Sections from the Distal colon were stored in RNAlater™(Qiagen, Valencia, Calif.) at −20° C. until RNA was isolated. RNA wasisolated using a RNeasy® Microarray Tissue kit and cDNA was generatedwith Taqman® Fast Advanced Master Mix. Tissue sections were scanned onan Aperio Scan Scope CS for analysis. Gene expression was analyzed witha Taqman® array FAST plate and read with an Applied Biosystems 7500 FastReal-Time PCR System.

Protein Levels. Sections from the Distal colon were flash frozen andstored at −80° C. until the protein was isolated. Tissue was pulverizedwith a Besseman Tissue Pulverizer and placed in T-PER with a proteaseinhibitor cocktail. Gravity centrifugation removed the debris and thesupernatant was collected and stored at 80° C. until analysis with anEMD Millipore mouse cytokine/chemokine magnetic bead panel.

Statistics. Summary data are means±SEM. Student T Test or ANOVA withBonferroni's post-hoc test were used for the determination ofstatistical significance among treatment groups, as appropriate.

As illustrated in FIGS. 13A-13C, oral administration of CTBp bluntedinjury in acute ulcerative colitis. While DSS exposure resulted insignificant weight loss in mice, the weight loss was blunted by CTBppretreatment (FIG. 13A). Additionally, CTBp decreased inflammation inthe tissue (approximately 1.7) compared to PBS+DSS group (approximately2.5) (FIGS. 13B and 13C). Referring to FIG. 14, oral administration ofCTBp blunted the disease activity index (DAI) score of mice exposed toDSS. The administration of CTBp significantly blunted the DAI scoreimmediately following the first dose and throughout the third DSSexposure period. The DAI is composed of body weight, fecal consistency,and occult blood.

As illustrated in FIGS. 15A-15C, oral administration of CTBp alsoblunted the tumor numbers (FIG. 15A) and tumor grades (FIG. 15B) of miceexposed to DSS. Tumor numbers per mouse were significantly decreased by3 μg CTBp and the tumor grades were significantly decreased by 3 and 10μg CTBp. Additionally, a shift to lower tumor score (FIG. 15C) andnumbers were noted with 3 and 10 μg CTBp. Tumor grades are shown in FIG.16, and include grade 1—small but detectable; grade 2—covers up to118^(th) of colon; grade 3—covers up to ¼ of colon; grade 4—covers up to½ of colon; and grade 5—covers over half of colon.

Oral administration of CTBp also significantly altered gene expressionin mice exposed to DSS (FIG. 17). For example, 3 μg CTBp administrationsignificantly increased FoxP3 expression following DSS exposure.Additionally, IFNγ expression trended to significance and Mapk8expression was significantly increased by 10 μg CTBp compared to PBSafter DSS exposure. Furthermore, as illustrated in FIG. 18, proteinlevels of tumor promoting cytokines (GM-CSF and IL-1α) were blunted byCTBp administration in DSS exposed mice. 3 and 10 μg CTBp significantlyblunted IL-1α protein levels following DSS exposure compared to PBS.MIP-2 was significantly blunted by 3 μg CTBp compared to PBS after DSSexposure. Interestingly, Interferon-γ, a tumor suppressor, wassignificantly elevated by CTBp administration. Gene expression analysisis currently underway to reveal additional protective factor(s).

In summary, oral administration of CTBp blunted CRC in the colons ofmice. Additionally, oral administration of CTBp after induction ofcolitis blunted colon tumor development in mice. The protein may bedeveloped as a novel oral immunotherapeutic agent against colitis and/orCRC.

Example 11—Plant-Made Cholera Toxin B Subunit as a Candidate OralImmunotherapeutic Agent against Ulcerative Colitis

The anti-inflammatory potential of orally administered CTBp in a mousemodel of ulcerative colitis was investigated. Briefly, C57BL/6J micewere exposed to dextran sodium sulfate (DSS) in drinking water for 7days and allowed to recover for 2 or 7 days before sacrifice. Mice wereorally administered twice with varying amounts of CTBp, dosed prior toor after the initiation of DSS exposure (prophylactic and therapeuticregimens, respectively). Upon sacrifice, body weights and fecal sampleswere analyzed for a Disease Activity Index (DAI). Colon was isolated andanalyzed for inflammatory gene expression by quantitative PCR andhistopathological scoring. Timing and dosage were selected based uponthe information presented in FIGS. 19-20, respectively.

Oral administration of CTBp significantly decreased the DAI, colonshrinkage, and histopathological scores. The maximum effect was observedwhen therapeutically dosed on Day 3 and 6, although the prophylacticregimen was also effective with higher dosages. The most effective doseof CTBp under the therapeutic regimen was determined to be 1-3 μg/mouse.Gene expression analysis revealed that CTBp significantly blunted theexpression of inflammatory cytokines in the colon, including interleukin(IL)-1β, IL-6 and IL-33.

As illustrated in FIGS. 21 and 22, CTBp administration increased theinnate immune cell populations in the colon without producing notableeffects on adaptive immune cell populations. Additionally, referring toFIGS. 23 and 24, various extracellular matrix components and remodelingenzymes were significantly increased by CTBp administration.Furthermore, as in Example 1, CTBp mitigated DSS-induced acute colitis(FIG. 25), prevented colitis-associated colon cancer development (FIG.26, and facilitated wound healing (FIGS. 27-28).

The foregoing data demonstrated that oral administration of CTBp hastherapeutic effects in chemically induced acute colitis in mice. Morespecifically, CTBp significantly altered the gene expression patterns inthe GI tract, with higher impacts in the colon than in the smallintestine and was found to be a mucosal wound healing molecule.Additionally, oral administration of CTBp mitigated chemically inducedacute colitis in mice, in part by increasing epithelial barrierfunctions and/or wound healing. Oral administration of CTBp also reducedtumorigenesis in a mouse model of colitis-associated colon cancer.Furthermore, oral administration of CTBp did not change the overallcomposition of gut microbiota.

Example 12—A Plant-Produced Cholera Toxin B Subunit Prevents AcuteColitis in a Mouse Model

Whether CTBp could also protect against acute colitis induced by dextransulfate sodium (DSS) was also investigated. Briefly, a wellcharacterized model of Ulcerative Colitis (UC) in mice is the DSS model,which induces a similar response as human UC (T helper 2 mediated).6C57BL/6J mice were orally administered 30 μg pCTB 2 weeks before and theday of initiation of DSS exposure. Mice were exposed to 4% DSS water adlibitum for eight days and allowed to recover for six days beforesacrifice.

Animals and treatments. Six week old female C57BL/6J mice were obtainedfrom Jackson Laboratory (Bar Harbor, Me.). For the DSS experiment,sodium bicarbonate was administered orally to neutralize the stomach pHprior to oral (gavage) doses of pCTB (30 μg) or PBS, which wasadministered two weeks prior to and the day of the initiation of DSSexposure. 4% DSS was administered for up to eight-days, following whichmice were put on laboratory water for up to a seven-day recovery period.Mice were sacrificed by CO₂ asphyxiation after the recovery period.

Percent Body Weight Change. Initial body weights were collectedimmediately prior to the initiation of DSS exposure. The body weightswere collected daily at similar times and percent change from baselinewas calculated. Percent change body weight without DSS exposure is shownin FIG. 29 and percent change in body weight with exposure to DSS isshown in FIG. 30. There were no significant changes in body weightfollowing administration of 30 μg pCTB as compared to PBS without DSSexposure (FIG. 29). While exposure to 4% DSS resulted in approximately10% body weight loss, pCTB showed significant protection whenadministered prior to DSS exposure by increasing body weight during therecovery phase compared to DSS alone (FIG. 30).

Hematoxylin and Eosin Staining. Tissue sections were collected from thedistal colon and placed in 10% formalin for 18 H. The tissue was thenplaced in 70% ethanol until the time of paraffin embedding. Paraffinembedding, cutting and H&E staining were performed by a trainedprofessional. Tissue sections were scanned on an Aperio Scan Scope CSfor analysis. Representative composite photomicrographs of tissuesections without exposure to DSS are illustrated in FIG. 31. Without DSSexposure, pCTB administration resulted in no morphological changes inmouse colons. Representative composite photomicrographs depicting PBS,PBS+DSS, and 30 μg pCTB+DSS are illustrated in FIG. 32. As shown in FIG.32, DSS exposure resulted in loss of epithelial integrity, increasedneutrophil infiltration, and ulceration, while CTB pre-treatmentdecreased the inflammatory injury seen in the DSS mice.

Masson's Trichrome Stain. Tissue sections were collected from the distalcolon and placed in 10% formalin for 18 H. The tissue was then placed in70% Ethanol until the time of paraffin embedding. Paraffin embedding andcutting were performed by a trained professional. Components of theTrichrome Stain were purchased from Electron Microscopy Sciences. Tissuesection ere scanned on an Aperio Scan Scope CS for analysis.Representative composite photomicrographs depicting PBS, PBS+DSS, andpCTB+DSS are shown in FIG. 33. DSS exposure resulted in increasedcollagen deposition and loss of epithelial integrity while 30 μg pCTBblunted collagen deposition and protected epithelial integrity followingDSS exposure, as compared to PBS+DSS.

Statistics. Summary data are means+/−SEM. ANOVA with Bonferroni'spost-hoc test Mann-Whitney rank sum test was used for the determinationof statistical significance among treatment groups, as appropriate.

CTBp Production in N. benthamiana. Tobamoviral vectors were vacuuminfiltrated into N. benthamiana leaves. After 5 days in a growth room,leaves were harvested and CTBp was purified.

Upon analysis of the results from the experiments, it was observed thatbody weights decreased in mice exposed to 4% DSS in drinking water. Thisweight loss was significantly blunted by CTBp pretreatment.Additionally, histologically scored inflammation in the colon wassignificantly decreased (FIG. 33). More specifically, DSS exposureresulted in a score of approximately 2.5 while CTBp pre-treatmentsignificantly decreased the inflammatory score (approximately 1.6) seenin the DSS mice. Massons' Trichrome staining revealed decreased fibrosisin the CTBp-treated mice. Immunohistochemical analysis suggested adecreased T cell population in the inflamed tissue by CTBp pretreatment.

In summary, administering CTBp prior to exposure to 4% DSS protectedmice from developing severe colitis. Additionally, pretreatment withCTBp protected against DSS-induced acute colitis by preventinginflammation and aberrant tissue regeneration. Furthermore, CTBpadministration protected mice from DSS-associated weight loss; decreasedinflammation in mice exposed to 4% DSS for 8 days; and completelyblunted fibrosis in mice exposed to 4% DSS.

Example 13—An Acute Mucosal Injury/Colitis Study in C57b1/6 Mice

Referring to FIG. 35, colonic injury/inflammation was induced by 3%(w/v) dextran sodium sulfate (DSS) in drinking water on Day 0-7. PBS, 1μg CTBp, or 1 μg CTB were orally administered on Day 3 and 6. On Day 9,Disease Activity Index (DAI; Qualis et al. Inflamm Bowel Dis 2009;15:236-247) was used to evaluate the therapeutic effect of CTB and CTBp.As illustrated in FIG. 36, CTBp provided decreased DAI as compared toCTB as well as PBS alone.

Materials and Methods for Examples 14-19

The following studies describe the impacts of orally administered CTB onthe GI tract in detail. As described below, a variant of CTB produced inNicotiana benthamiana plants (CTBp; SEQ ID NOS: 3 and 4) was utilized,because the protein can be efficiently manufactured at scale whileshowing GM1 ganglioside-binding affinity, physicochemical stability andoral immunogenicity for anti-toxin antibody induction comparable tooriginal CTB. Thus, CTBp provided a viable alternative to the vaccineantigen included in Dukoral® and potentially facilitates other clinicalapplications as described above. The global impacts of CTBp oraladministration on the small intestine and colon were first characterizedby elucidating changes in their immune cell populations and geneexpression profiles. The results led to the hypothesis that CTBp mightinduce TGFβ-mediated mucosal wound healing, which was subsequentlydemonstrated in an in vitro human colon epithelial model using the Caco2cell line. To examine the clinical relevance of these findings, adextran sulfate sodium (DSS) mouse model of intestinal wounding andulcerative colitis was employed, another major form of inflammatorybowel disease (IBD) along with Crohn's disease, to which CTB'sinfluences have not been reported before. Furthermore, as mucosalhealing potentially lowered colorectal cancer risk in ulcerativecolitis, the effect of CTBp treatment on ulcerative colitis-associatedtumorigenesis was investigated in the azoxymethane (AOM)/DSS model. Thedata point to the potential utility of CTBp as an oral therapy forulcerative colitis in addition to mass vaccination against cholera.

Animals. Eight-week-old C57BL/6J female mice were obtained from JacksonLaboratories (Bar Harbor, Me.). Animal studies were approved by theUniversity of Louisville's Institutional Animal Care and Use Committee.

Study design. For all animal experiments, five to nine mice per group,randomly assigned, were used. For the characterization of the globalimpacts of CTBp oral administration, animals were gavaged with PBS or 30μg CTBp twice at a 2-week interval after neutralization of stomach acidswith a sodium bicarbonate solution, as described previously. CTBp wasproduced in N. benthamiana and purified to >95% homogeneity with anendotoxin level of <1 endotoxin units/mg, as described previously. Twoweeks after the second dose, mice were sacrificed, and feces, colon,small intestine, spleen and Peyer's patches were collected. For theacute DSS “vaccination” study, animals were orally administered with PBSor 30 μg CTBp as described above. DSS exposure was initiated on the dayof the second dosing (FIG. 50), using a method slightly modified from apreviously published protocol. Body weights were measured at theinitiation of DSS exposure as a baseline and every morning thereafter todetermine percent change. To determine DAI, animals were scored on adaily basis with the scoring rubric adapted from the literature. Animalsreceived 4% DSS (M.W. 36,000 to 50,000; MP Biomedicals, Santa Ana,Calif.) in drinking water for 8 days, and allowed to recover 6 daysduring which the animals received normal drinking water. For thetherapeutic dose ranging study, mice were orally administered with PBSor 0.1-30 μg CTBp twice (day 3 and 6) during DSS exposure. Animals wereexposed to 3% DSS for 7 days and allowed 2-day recovery. For the AOM/DSSstudy (FIG. 51), AOM (10 mg/kg) was administered by intraperitonealinjection. DSS exposure (2%) was initiated 1 week after the AOMinjection for 7 days and allowed to recover for 14 days. The DSSexposure and recovery cycle was repeated 3 times and mice weresacrificed following the 3^(rd) cycle.

Immune cell isolation. Lamina propria lymphocytes were isolated from thecolons and small intestines by a series of washing and collagenasedigestion steps. Epithelial cells, mucus and fat tissue were removed byincubating with EDTA at 37° C. The intestinal tissues were cut intosmall pieces and incubated with collagenase at 37° C. Splenocytes wereisolated by crushing the spleens on metal mesh and separating thesupernatant. An ammonium chloride potassium carbonate buffer was used tolyse red blood cells and following several washes the cells werefiltered through a 70 μm cell strainer. Peyer's patch lymphocytes wereisolated by chopping up the Peyer's patches with fine surgical scissorsand incubating the pieces in collagenase at 37° C. The collagenase stepwas repeated and the second suspension was isolated. After a second washthe cells were combined and filtered through a 70 μm cell strainer.

Flow cytometry. Immune cells from 2 mice were pooled for each biologicalreplicate with a total of at least 4 biological replicates per group.Cells were stained using appropriate antibodies and a Cell Staining kitfrom eBiosciences, Inc. (San Diego, Calif.). Briefly, tubes containing1×10⁶ cells were washed with Flow Cytometry Staining Buffer (FCSB;supplied in the kit) 2 times. Fc Block was added to each tube in FCSBfor 10 min. For CD4 cell populations, cells were incubated with surfacestaining antibodies (anti-CD3-FITC, anti-CD4-APC-Cy7, anti-CD25-PerCP)at 4° C. for 30 min. After removing excess antibodies,Fixation/Permeabilization Buffer (FPB; supplied in the kit) was addedand incubated overnight. Cells were washed with Permeabilization Buffer(PB, supplied in the kit) and again incubated for 10 min with Fc block.Internal cell antibodies (Gata3-PE, T-Bet-PE-Cy7, FoxP3-APC,IL-17-eFlour450) were then added and incubated for 30 min at 4° C.Finally, cells were washed and suspended in FCSB. For other immune cellpopulations, cells were incubated with surface staining antibodies(CD19-APC, CD3-FITC, CD49b-PE, F4/80-PeCy7, CD11c-PerCP-Cy5.5,CD8-APC-eFluor 780, and CD45-eFlour450) at 4° C. for 30 minutes. Afterremoving excess antibodies, FPB was added to the tubes and incubatedovernight. Cells were then washed and resuspended in FCSB. Events(1×10⁵) were counted on a BD FACSCanto™ II and analyzed with the BDFACSDiva Software v6.1.3.

RNA isolation. Sections from the small intestine and distal colon werestored in RNAlater™ (Qiagen, Valencia, Calif.) at −20° C. until RNA wasisolated. Colon tissue (approximately 14 mg) was placed in QIAzol lysisreagent in a 2.0 mL conical bottom centrifuge tube with Zirconia/Silicabeads. A Bead Bug™ (Denville Scientific Inc., Mass.) was used tohomogenize the tissue. An RNeasy® Microarray Tissue Kit from Qiagen wasused to purify the RNA from the tissue homogenate. RNA was stored at−80° C. until use.

Microarray gene expression analysis. Total RNA was amplified and labeledfollowing the Affymetrix (Santa Clara, Calif.) standard protocol forwhole transcript expression analysis, followed by hybridization toAffymetrix Mouse Gene 2.0 ST® arrays. The arrays were processedfollowing the manufacturer recommended wash and stain protocol on anAffymetrix FS-450 fluidics station and scanned on an AffymetrixGeneChip® 7G scanner using Command Console 3.3. The resulting .cel fileswere imported into Partek Genomics Suite 6.6 and transcripts werenormalized at the gene level using RMA as normalization and backgroundcorrection method. Contrasts in a one-way ANOVA were set up to comparethe treatments of interest.

qRT-PCR. First strand cDNA was obtained from reverse transcription of150 ng RNA using a SUPERSCRIPT VILO cDNA synthesis kit (LifeTechnologies) according to the manufacturer's instructions. TemplatecDNA were added to a reaction mixture containing 10 μl of 2× TaqMan®Fast Advanced Master Mix (Life Technologies) and endonuclease free waterto 20 μl and loaded in TaqMan® Array Standard 96 well Plates (AppliedBiosystems). These plates contain pre-spotted individual TaqMan® GeneExpression probes for the detection of genes of interest as well as thehouse keeping genes 18S, β-actin (ACTB), and GAPDH (Table 1). PCRamplification was carried out on a 7900HT Fast Real-Time PCR System(Applied Biosystems) with the following conditions: 95° C., 20 min; 40cycles (95° C., 1 min); 20 min at 60° C. The 7500 Software v2.0.6(Applied Biosystems) was used to determine the cycle threshold (Ct) foreach reaction and derive the expression ratios relative to control.Wound healing pathway analysis was performed with a RT2 Profiler PCRMouse Wound Healing Array (Qiagen) under the same conditions describedabove.

TABLE 1 Gene Identity for qRT-PCR analysis. Gene Entrez Gene Name IDGene ID Angiogenin, ribonuclease A family, member 4 Ang4 219033Angiopoietin 1 Angpt1 11600 ATP-binding cassette, sub-family A (ABC1),Abca1 11303 member 1 Cathepsin K Ctsk 13038 Collagen, type 1, alpha 1Col1a1 12842 Collagen, type 1, alpha 2 Col1a2 12843 Collagen, type 3,alpha 1 Col3a1 12825 Collagen, type XIV, alpha 1 Col14a1 12818 Colonystimulating factor 2 (granulocyte- Csf2 12981 macrophage) Decorin Dcn13179 Interleukin 1 beta Il1b 16176 Interleukin 33 Il33 77125 Malatedehydrogenase 1, NAD Mdh1 17449 Matrix Metallopeptidase 2 Mmp2 17390Mitogen-activated protein kinase kinase kinase Map4k4 26921 kinase 4 NLRfamily, pyrin domain containing 3 Nlrp3 216799 SMAD family member 6Smad6 17130 Tissue inhibitor of metalloproteinase 4 Timp4 110595Transforming growth factor, beta 1 Tgfb1 21803 Transgelin Tagln 21345Tumor necrosis factor Tnf 21926

Caco2 wound healing assay. The Caco2 wound healing assay was performedusing a modified method. Briefly, the cells were seeded and grown toconfluence in 6 well plates (Thermo Scientific™ Nunc™ Cell-CultureTreated). The culture medium was discarded, two 0.5-1.0 mm across linearwounds were made per well with a 200 μL sterile beveled pipette tip (USAScientific) and cells were washed with PBS to remove loose cells. PBS,CTBp (0.3-3 μM), TGFβ1 (0.2 nM), and/or an anti-TGFβ1,2,3 antibody (3.85nM; Abcam) were subsequently added in fresh serum-deprived medium.Photomicrographs of the wounds were taken 0, 24 and 48 h after thewounding at 4× magnification. Quantification of the remaining cell-freearea to the initial wound area was measured using the public domainsoftware Image J and calculated as a mean percentage per well. Theculture medium/supernatants were collected from each well 48 h afterwounding and stored −80° C. until analysis. The culture supernatantswere analyzed by a human Cytokine/Chemokine or TGFβ1,2,3 Magnetic BeadPanel (EMD Millipore). The panel was analyzed with a Milliplex MAP Kiton a MagPix with Luminex xMAP technology.

Immunohistochemistry. Colons were removed and washed with PBS. A portionof the distal colon was fixed with paraformaldehyde overnight and storedin 70% ethanol until paraffin embedding and sectioning. Sections weredeparaffinized with Citrisolv and rehydrated through several ethanolwashing steps ending with incubation in distilled water. Antigenretrieval was performed overnight with a 2100 Retriever (ElectronMicroscopy Sciences) using Buffer B designed specifically for theRetriever. Tissue sections were blocked for endogenous peroxidase,avidin, biotin, and serum from the animal in which the secondaryantibody was raised. Primary antibody (anti-F4/80; Abcam) was incubatedwith the tissue sections for 2 h at room temperature. The VectastainElite ABC kit (rabbit anti-goat; Vector Labs) was used to label theprimary antibody. F4/80+ cells were visualized with the ImmPACT DABSubstrate Kit (Vector Labs) and then dehydrated through an ethanolgradient and finally incubated with Citrisolv. Sections were scannedusing a Aperio ScanScope CS (Leica Biosystems) and positive cells werecounted, in a blinded manner, in 10 representative sections (40×magnification) from each colon. The 10 sections were averaged and thatwas the score for each animal.

Histology. Colons were removed and washed with PBS. A portion of thedistal colon was fixed with paraformaldehyde overnight and stored in 70%ethanol until paraffin embedding, sectioning and routine H&E staining.Inflammation scoring was performed using a scale that has beenpreviously published. Tissue sections from 8 mice were scored in ablinded manner and averaged for each group. Masson's Trichrome Stain wasperformed using a kit purchased from Electron Microscopy Sciences(Masson's Trichrome for Connective Tissues).

Protein isolation and quantification. Distal colon tissue isolated atsacrifice was snap frozen in liquid nitrogen and pulverized with aBessman Tissue Pulverizer and placed in T-PER (Thermo Scientific) with aprotease inhibitor cocktail (Sigma-Aldrich). Total protein was isolatedby gravity centrifugation of tissue fragments followed by collection ofthe buffer containing isolated protein, and storage at −80° C. untilanalysis. Protein sample concentrations were determined using a Nanodrop1000 (Thermo Scientific). Protein was normalized for all samples priorto loading on a Mouse Cytokine/Chemokine Magnetic Bead Panel (EMDMillipore). The panel was analyzed with a Milliplex MAP Kit on a MagPixwith Luminex xMAP technology.

Tumor scoring. Tumors were scored via endoscopic analysis of the fulllength of the colon. Tumor scoring was based on the following rubric: 0for no tumor, 1 is a very small but detectable tumor, 2 the tumor coversup to ⅛ colon circumference, 3 tumor covers ¼ of colon circumference, 4tumor covers up to ½ of colon, and 5 tumor covers more than ½ of colon(FIG. 43B).

Microbiome analysis. Fecal samples were collected at the end of theacclimation period and at the time of study termination. Bacterial DNAwas isolated using the PowerFecal® DNA Isolation Kit (Mo BioLaboratories, Inc.). Briefly, fecal samples were added to a bead tubewith solution and lysed with a bead beater. Through a series ofcentrifugation and elution steps Fecal DNA was isolated. DNAconcentration was determined using the Quant-iT dsDNA Broad-Range Kit(Life Technologies). Samples were then sent to Second Genome, Inc. foranalysis. Upon arrival, samples were enriched for bacterial 16S V4 rDNAregion by utilizing fuxion primers designed against conserved regionsand tailed with sequences to incorporate Illumina flow cell adapters andindexing barcodes. Amplified products were concentrated using asolid-phase reversible immobilization method and quantified byelectrophoresis using an Agilent 2100 Bioanalyzer®. Samples were loadedinto a MiSeq® reagent cartridge and then loaded into the instrument.Amplicons were sequenced for 250 cycles with the MiSeq instrument.Second Genome's PhyCA-Stats™ analysis software package was used toanalyze the results.

Statistics. For all data, outliers were determined by statisticalanalysis using the Grubb's test (P<0.05) and excluded from furtheranalysis. Graphs were prepared and analyzed using Graphpad Prism version5.0 (Graphpad Software, La Jolla, Calif.). To compare two data sets, anunpaired, two-tailed Student's t test was used. To compare three or moredata sets, one-way ANOVA with Bonferroni's multiple-comparison post-testor Kruskal-Wallis test with Dunn's multiple-comparison post-test wereperformed. For body weights and DAI results, a two-way ANOVA withBonferroni's multiple-comparison post-test was employed.

Example 14—Colon Lamina Propria Leukocyte Profile is SignificantlyAltered by CTBp Oral Administration.

Using flow cytometry, the immune cell populations of the lamina propriaof small intestine and colon, Peyer's patches, and spleen wascharacterized in mice two weeks after CTBp oral administration. Theanalysis revealed that subsets of innate immune cell populations in thelamina propria of colon (FIG. 37), but not of the small intestine (FIG.44), significantly increased; macrophages (F4/80+), dendritic cells(DCs; CD11c+) and natural killer (NK) cells (CD49b+) were significantlyincreased when compared to the control PBS group (FIG. 37A). Theincrease of these cell types was associated with a relative decrease inB cells within the CD45+ cell population (FIG. 37B). Increasedmacrophage infiltration into colon lamina propria upon CTBpadministration was confirmed by immunohistochemistry analysis (FIG.37C). Despite the significant increase of macrophages, there was noabnormality or inflammation noted in the colon mucosa. Meanwhile, such amajor shift in immune cell profiles was not observed in the smallintestine lamina propria, Peyer's patches or spleen (FIG. 44),indicating compartmentalized impacts of orally administered CTBp onimmune cells in different regions of the GI tract.

Example 15—CTBp Oral Administration has a More Pronounced Effect onColon Gene Expression than Small Intestine

To further evaluate the impacts of orally administered CTBp on the GItract, microarray analysis of transcripts isolated from the smallintestine and colon was performed . The protein had profound impacts onthe gene expression profile of both upper and lower GI tract (FIG.38A-38C and FIG. 45). However, while gene expression profiles in smallintestine samples clustered relatively tightly, colons from CTBp-treatedmice showed a completely separated pattern compared with the controlsamples (FIG. 38A). At a global level, 871 genes were significantlyaltered in the colon between CTBp and PBS groups (P<0.01; one-wayANOVA), while ˜5 fold less (i.e., 184) genes were significantly alteredin the small intestine (FIG. 38B). Of these significantly altered genes,539 were induced and 332 were suppressed in the colon. By comparison,the small intestine was fairly evenly split between induced andsuppressed genes, with 97 and 87 altered genes, respectively, and therewas no overlap with genes affected in the colon.

Example 16—CTBp Enhances TGFβ-Associated Gene Expression Pathways in theColon

A gene expression pathway analysis (MetaCore™ version 6.22 build 67265)revealed that extracellular matrix (ECM) remodeling and epithelial tomesenchymal transition (EMT) pathways were among the most significantlyinduced pathways in the colon upon CTBp oral administration. Inparticular, TGFβ-dependent pathways heavily populated the inducedpathways (FIG. 38C). Indeed, when evaluating individual gene expressionfrom the microarray analysis Tgfb1, TgfbII receptor and Smad4 were foundto be significantly induced by CTBp (FIG. 46), which is indicative ofepithelial wound healing activity. By contrast, such strong induction ofTGFβ-related pathways was not observed in the small intestine.Suppressed pathways in the colon epithelium included several metabolicpathways, cystic fibrosis transmembrane conductance regulator (CFTR)pathways, and an apoptosis associated pathway. Genes associated withlipid, bile acid, pyruvate, and androstenedione and testosteronemetabolic pathways were significantly blunted by CTBp. To confirmmicroarray data, quantitative real-time reverse-transcription PCR(qRT-PCR) was performed on selected induced, suppressed, or unalteredgenes; a high agreement between microarray and qRT-PCR results wasobtained (FIG. 47). Notably, a wound healing pathway-focused qRT-PCRanalysis revealed that many key genes, including Col1a1, Col1a2, Col3a1,Col14a, Mmp2, Ctsk, Tagln and Angptl, were significantly upregulated byCTBp oral administration.

Example 17—CTBp Enhances Wound Healing in Human Colonic Epithelial Cells

To investigate the mucosal wound healing potential of CTBp suggested bythe gene expression analysis, the human colon epithelial cell line Caco2wound healing model 14 was employed. As shown in FIGS. 39A-39B, CTBp(1.0 and 3.0 μM) significantly enhanced wound closure, similarly to theTGFβ1 control. Indeed, increased levels of TGFβ1 and TGFβ2 were noted 24h post wounding in the culture supernatant of CTBp treated cells(Supplementary FIG. 49), and co-incubation of CTBp with ananti-TGFβ1,2,3 neutralizing antibody completely inhibited the woundhealing activity (FIG. 39C). Analysis of inflammation and woundhealing-related cytokines in the culture supernatant at 48 h revealedthat CTBp and TGFβ1 had an overall similar cytokine profile (FIG. 39D);both produced similar levels of epidermal growth factor (EGF),fibroblast growth factor (FGF)-2, granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),interferon (IFN)-β, interleukin (IL)-10, IL-1β, IL-5, IL-6, tumornecrosis factor (TNF)α and vascular endothelial growth factor (VEGF), incontrast to the PBS control. An exception was monocyte chemoattractantprotein (MCP)-1, which was significantly elevated only in TGFβ1-treatedcells.

Example 18—CTBp Mitigates DSS-Induced Acute Colonic Injury andInflammation

It was next determined if the in vitro mucosal healing activity of CTBpcould be translated into a therapeutic effect in vivo. Awell-established mouse DSS colitis model was employed, which inducesinjury and severe inflammation in the distal colon. In an initial study,the “vaccination” regimen that brought about the above-describedbiological effects implicated in mucosal protection was used (FIGS.37-38); PBS or 30 μg CTBp was orally administered twice at a 2 weekinterval to mice prior to DSS exposure (FIG. 50 7). As shown in FIG.40A, CTBp significantly blunted the weight loss induced by DSS.Histopathological examination on hematoxylin and eosin (H&E)-staineddistal colon tissue at 6 days post DSS exposure revealed that CTBptreatment prevented the aberrant loss of crypts and ulceration that werenoted in the untreated control group; although shortening of basalcrypts and mild inflammatory infiltrates were observed, the epithelialsurface remained intact (FIG. 40B-40C). Despite the significantly lessdamage and inflammation in the colon of CTBp-administered mice, thenumbers of macrophages infiltrated into the colon mucosa were similarbetween CTBp-treated and untreated groups, and significantly higher thanthat of the non-DSS-exposed control group (FIG. 40D-40E). Moreover, CTBpadministration appeared to prevent fibrosis in the colon according toMasson's trichrome stain, while fibrosis was evident in the DSS-exposedvehicle control-administered group (FIG. 40F). Consistent with this, themajor fibrotic genes Col1a1 and Tgfb116, 17 were significantly increasedin the colon tissue of the DSS-exposed vehicle control-administeredmice; CTBp treatment, on the other hand, showed lower levels of thesetranscripts (FIG. 40G).

Given that CTBp significantly improved recovery from DSS-induced acutecolitis, its effect immediately after DSS exposure was alsoinvestigated. At this maximum injury/inflammatory point, CTBpadministration again significantly reduced the mucosal damage,characterized by shortened yet visible basal crypts, relatively mildinflammatory infiltrates in the mucosa and submucosa, and retention ofthe epithelial cell surface unlike the PBS control group. Meanwhile,daily oral administration of 100 μg mesalamine (MES) during the DSSexposure, which simulates a current treatment for ulcerative colitis inhumans, showed similar protection observed with the CTBp regimenemployed here (FIG. 41A). To further characterize CTBp's protectiveeffect, qRT-PCR analysis was performed. CTBp treatment was shown toblunt the escalation of representative inflammatory marker expressioninduced by DSS exposure, including Il1b, Il33, Il6 and Infg (FIG. 41B).Tgfbl, on the other hand, showed a significant increase in bothCTBp-treated and vehicle control-administered mice following DSSexposure compared to healthy animals. Analysis of soluble inflammatorymarkers in the distal colon also showed that CTBp administration bluntedthe significant increase of major inflammatory proteins, includingIL-1β, IL-3, IL-4, IL-5, IFNγ, IL-6 and GM-CSF (FIG. 41C). Of note, CTBpadministration did not increase IL-10 either at the gene expression orprotein levels, despite that IL-10 has previously been linked to thepotential anti-inflammatory activity of CTB and CT.

Since CTBp “vaccination” was effective in DSS-induced acute colitis, wenext examined the protein's therapeutic dosing effect. As shown in FIGS.42A-42D, protection was evident as CTBp was dosed at the late phase ofthe DSS exposure (Day 3 and 6), when the onset of colonic epithelialdamage had already taken place. This demonstrates that the protein doesnot require pre-emptive dosing for protection against DSS-induced colonepithelial insult. Conversely, CTBp's mucosal protective activity cantake effect relatively quickly against ongoing epithelial damage andinflammation. A dose-ranging study (0.1-30 μg/mouse/dose) showed thatthe most effective dose of CTBp in this therapeutic regimen was as lowas 1 μg/mouse/dose. Interestingly, the efficacy did not follow adose-dependent pattern; 3, 10 and 30 μg were less effective than 1 μg,according to disease activity index (DAI) scores. Nevertheless, thehighest dose still prevented the shortening of the colon length, showeda significantly lower inflammation score than the untreated control, andreduced epithelial damage and ulceration in the distal colon tissue,suggesting no adverse effects at this dose (FIGS. 42A-42D).

Example 19—CTBp Oral Administration can Protect Against Chronic Colitisand Colon Tumor Development in an AOM/DSS Model

The significant protection seen in the acute colitis/colon injury modelprompted an investigation into whether CTBp could also be effective inan AOM/DSS mouse model of chronic colitis and colon cancer. CTBp (3 or10 μg) was given at the end of the first DSS exposure period followed bythree additional doses every two weeks for a total of four doses (seeFIG. 51). As shown in FIGS. 43A-43F, CTBp administration (3 μg)significantly decreased the DAI score immediately following the firstdose and more dramatically during the 3rd DSS exposure period (FIG.43A). Such a clear effect was not observed with the higher dose (10 μg)of CTBp. However, tumorigenesis was significantly reduced at both doselevels (FIG. 43C).

Tumor growth also appeared to be limited by CTBp, as indicated by thedecreased number of grade 3-5 tumors and total tumor grade inCTBp-administered groups (FIGS. 43C-43D). Of note, CTBp administration(10 μg) without DSS exposure did not induce any sign of intestinaldamage or tumor growth (FIG. 43A, 43C, 43D).

To further characterize the protection of 3 μg CTBp in the AOM/DSSmodel, markers associated with inflammation and cancer development wereevaluated in the colon tissue two weeks following the final DSSexposure. Both DSS-exposed groups showed an increased level of Infg,Tgfb1, Foxp3, Il10, Nlrp3, Csf2 and Tnfa. However, CTBp administrationresulted in a more significant increase in Tgfb1, Foxp3 and Ifngcompared to healthy mice (FIG. 43E). Analysis of soluble markers showedthat, although IL-1β had declined to a baseline level at this point,several inflammatory markers still showed a significantly higher levelin the DSS-exposed, vehicle control-administered group compared tohealthy mice, including TNFα, IL-1α, keratinocyte chemoattractant (KC),chemokine (C-X-C motif) ligand 9 (CXCL9) (FIG. 43F). CTBp administrationblunted the elevation of these markers, most notably IL-1α, which is akey factor to exacerbate gut inflammation. Notably, CTBp-treatmentcompletely blocked the AOM/DSS-induced decrease of IFNγ and IL-2 levels,which could be linked to the reduced tumorigenesis in this group.

Discussion of Examples 14-19.

Oral administration of CTB leads to a robust antibody response and ananti-inflammatory effect. The former represents the protein's mostwell-known biological activity, which has been exploited in choleraprevention (as a component of Dukoral® vaccine). On the other hand, theutilization of CTB's anti-inflammatory activity in inflammatory diseasetherapy is yet to be achieved, in part due to its obscure underlyingmechanisms. The present study revealed that oral administration of CTBpexhibits previously unidentified impacts on the distal part of the GItract; recruitment of macrophages, DCs and NK cells into the colonlamina propria and upregulation of TGFβ pathways in the colon (FIGS.37A-37C and FIGS. 38A-38C), which subsequently lead to the discoverythat CTBp promotes mucosal healing in the colon (FIGS. 39-43). The useof CTBp was justified because it retains key molecular features oforiginal CTB. Although CTB was previously shown to upregulate TGFβ inimmune cells and blunt intestinal inflammation of Crohn's disease, thepresent studies demonstrated for the first time that orally administeredCTB can facilitate TGFβ-driven mucosal wound healing in the colon. TGFβhas multifaceted functions including pivotal roles in gut homeostasisand intestinal wound healing. A recent study has shown that thesuppression of TGFβ signaling in an injured and inflamed mucosa leads toinvasive tumor development in the colon, showing that TGFβ-mediatedmucosal repair plays a key role in colitis-associated colon tumorprevention. This is in line with our findings in the AOM/DSS study (FIG.43). However, TGFβ1 is also known to be elevated in collagenous colitisand connected to increased collagen deposition and fibrosis, indicatingthe dual nature of this cytokine in mucosal remodeling. In this regard,the fact that CTBp administration prevented fibrosis in the acute DSScolitis study (FIGS. 40F-40G) suggests that the CTBp dosing regimen didnot overstimulate TGFβ signaling to a level causing adverse effects.Interestingly, the Caco2 study showed that CTBp did not dose-dependentlyinduce TGFβ1 or TGFβ2 (FIG. 49), which might represent feedbackinhibition by CTBp overdose. This may in turn explain why the higherdoses of CTBp were not as effective as the lower dose in the acute andchronic DSS studies (FIGS. 42-43). Detailed investigation into CTBp'sdose-effect relationship is warranted to define a therapeutic window foroptimal mucosal healing.

It is of interest to note that CTBp-induced TGFβ activation in the colonepithelium was accompanied by a significant increase of several innateimmune cells in the same mucosa (FIGS. 37-38). CTB is known to alter theT cell profile under various conditions and affect DC maturation invitro. However, the protein's impacts on immune cells in differentregions of the GI tract, as shown in the present study, areunprecedented. At this point, the mechanism by which CTBp induced suchcompartmentalized effects on GI tract immune cells is not clear. Apossible change in the gut microbiota was suspected. However, theoverall fecal microbiome profile showed no discernible shift at thepoint when colon gene expression and immune cell profile changes wereobserved (FIG. 52). Bacteroidetes and Firmicutes spp. dominated at thephylum level (FIGS. 53A-53B), which is typical for C57BL/6J mice. Therewere significant changes at the species level in 12 OperationalTaxonomic Units with 11 belonging to the Clostridiales order, but theseconstitute minor subpopulations in the gut flora (FIG. 53C). Theseresults suggested that the gut microbiota was unlikely the cause of thedrastic changes in the colon observed in the present study, althoughmore detailed investigation is warranted to reveal possible impacts ofCTBp on gut microbiota over a longer term. Meanwhile, the observationthat CTBp concomitantly stimulated the activation of TGFβ signaling andthe increase of the innate immune cells in the colon leads us topostulate that the latter effect might also play a role in the protein'smucosal healing effects. For example, lamina propria-resident DCs werepreviously shown to suppress the severity of DSS colitis. NK cells playa major role in tissue remodeling by clearing dead or dying cells.Macrophages can remove bacteria that penetrate the epithelium anddamaged tissue and play an important role in enhancing late phase woundhealing, which might explain our observation that CTBp markedlyincreased macrophage infiltration into the colon mucosa in DSS-exposedmice (FIG. 40D-40E) while significantly reducing epithelial damage (FIG.40B-40C). Studies are currently underway to elucidate the mechanism andthe potential contribution of CTBp-induced colonic immune cell profilein the context of mucosal healing.

In the mouse DSS model, CTBp oral administration significantly reducedulceration in DSS-exposed colon epithelia (FIGS. 40-42), which isindicative of enhanced mucosal healing. Additionally, the escalation ofinflammatory markers was blunted independently of the stronganti-inflammatory cytokine IL-10, while Tgfb1 expression significantlyincreased (FIG. 41B-41C). Coupled together, it was strongly suggestedthat CTBp's protective effect was primarily ascribed to theTGFβ-mediated mucosal healing activity demonstrated in the Caco2 woundhealing assay (FIG. 39). Since DSS colitis is pathologically similar toulcerative colitis in humans, the data point to a possibility that CTBpis effective against ulcerative colitis, a major form of IBD along withCrohn's disease. In particular, mucosal healing has recently become animportant target for ulcerative colitis therapy because it is associatedwith improved clinical outcomes. It should be noted that only two loworal doses of CTBp were as effective as mesalamine dosed daily duringthe 8-day DSS exposure in the acute colitis model (FIG. 41), andbiweekly dosing of CTBp also proved to be effective against chroniccolitis and reduced colitis-associated colon tumorigenesis (FIG. 43),highlighting the protein's remarkable therapeutic effect.

In summary, the foregoing work revealed a novel function of CTBp toenhance colonic mucosal healing through TGFβ pathways. Oraladministration of CTBp provided protection against DSS-induced colonicinjury and colitis. Although further investigations are required tocarefully define the dose-effect relationship in mucosal remodeling andefficacy in other clinically relevant conditions such as Smad7overexpression, an efficient bioproduction system already available forCTBp9 should significantly facilitate the protein's preclinical andclinical investigations towards its potential use to promote colonicmucosal health besides cholera prevention.

Example 20—Comparison of Plant-Produced N4S-CTB-SEKDEL (CTBp) withOriginal CTB in an Acute Colitis Mouse Model

The effects of therapeutic dosing of CTBp and CTB were investigated inC57b1/6 mice exposed to 3% dextran sodium sulfate (DSS) in drinkingwater for 7 days. At the end of DSS exposure, mice were orallyadministered with either 3 μg CTBp, 3 μg CTB, or PBS vehicle controlonce. Percent body weight change was monitored daily until sacrifice onday 14. As illustrated in FIGS. 54A-54C, the administration of CTBpsignificantly affected percent body weight change, colon length, anddisease activity index (DAI) as compared to both PBS and original CTB.More specifically, as illustrated in FIG. 54A, administration of CTBpprovided significant increases in percent body weight change as comparedto both PBS and CTB. Additionally, as illustrated in FIGS. 54B and 54C,respectively, administration of CTBp provided a significant increase incolon length and a significant decrease in disease activity index ascompared to both PBS and CTB. In contrast, there were no significantdifferences observed in colon length (FIG. 54B) or disease activityindex (FIG. 54C) between original CTB and PBS. Thus, in someembodiments, CTBp provides increased wound healing and/or diseasetreatment as compared to original CTB and/or PBS.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

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It will be understood that various details of the presently-disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A method of treating a disease, comprisingadministering to a subject in need thereof an effective amount of acomposition including a cholera toxin B subunit variant having one ormore modifications.
 2. The method of claim 1, wherein the one or moremodification to the cholera toxin B subunit variant increase theexpression of the polypeptide in a plant cell.
 3. The method of claim 1,wherein the one or more modification to the cholera toxin B subunitvariant include an Asn4 to Ser mutation.
 4. The method of claim 3,wherein the Asn4 to Ser mutation reduces N-glycosylation.
 5. The methodof claim 1, wherein the one or more modification to the cholera toxin Bsubunit variant includes a C-terminal hexapeptide sequence attached forendoplasmic reticulum (ER) retention.
 6. The method of claim 5, whereinthe one or more modification to the cholera toxin B subunit variantfacilitates recombinant production.
 7. The method of claim 1, whereinthe cholera toxin B subunit variant is substantially immunologicallyidentical to a cholera toxin B subunit.
 8. The method of claim 1,wherein the disorder is cancer.
 9. The method of claim 8, wherein thecancer is colon cancer.
 10. The method of claim 1, wherein the disorderis an inflammatory disorder.
 11. The method of claim 1, furthercomprising administering the composition to the subject withoutsubstantially changing a fecal microbiome of the subject.
 12. The methodof claim 1, wherein the administering of the composition includes oraladministration.
 13. The method of claim 1, wherein the administering ofthe composition decreases protein levels of tumor promoting cytokines.14. The method of claim 1, wherein the administering of the compositionincreases the innate immune cell populations in the subject's colon. 15.The method of claim 14, wherein the administering of the compositionincreases the innate immune cell populations without producingsubstantial effect on adaptive immune cell populations.
 16. The methodof claim 1, wherein the administering of the composition provides anincreased effect on colon gene expression as compared to small intestinegene expression.
 17. The method of claim 1, wherein the cholera toxin Bsubunit variant comprises the sequence of SEQ ID NO: 4, SEQ ID NO: 6, orSEQ ID NO:
 25. 18. The method of claim 1, wherein the one or moremodifications comprise a secretory signal peptide selected from thegroup consisting of a rice alpha-amylase secretory signal peptide, aNicotiana plumbagenifolia calreticulin secretory signal peptide, anapple pectinase secretory signal peptide, and a barley alpha-amylasesecretory signal peptide.
 19. The method of claim 1, wherein the one ormore modifications comprise a secretory signal peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOS: 18, 20,22, and
 24. 20. The method of claim 19, wherein the secretory signalpeptide comprises the rice alpha-amylase secretory signal peptide. 21.The method of claim 1, wherein the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 26-29. 22.The method of claim 1, wherein the one or more modifications comprise anendoplasmic reticulum retention signal having the amino acid sequenceKDEL.
 23. The method of claim 1, wherein the cholera toxin B subunitvariant includes two or more N-linked glycosylation sequons.
 24. Themethod of claim 23, wherein the cholera toxin B subunit variantcomprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, or SEQ ID NO:
 14. 25. The method of claim 1, wherein theinflammatory disorder is an inflammatory bowel disease.
 26. The methodof claim 25, wherein the inflammatory bowel disease is selected fromulcerative colitis and Crohn's disease.
 27. The method of claim 1,wherein the cancer is colitis-associated colon cancer,
 28. The method ofclaim 1, wherein the inflammatory disorder is a gastrointestinalinflammation and/or injury.
 29. The method of claim 28, wherein thegastrointestinal inflammation and/or injury is selected from celiacdisease, irritable bowel syndrome, radiation-induced colitis, andinfection-induced colitis.
 30. The method of claim 1, wherein theinflammatory disorder is a mucosal inflammation and/or injury.
 31. Themethod of claim 30, wherein the mucosal inflammation and/or injury isselected from asthma, airway burns, corneal injury, and vaginosis.
 32. Amethod of enhancing wound healing, comprising administering to a subjectin need thereof an effective amount of a composition including a choleratoxin B subunit variant having one or more modifications.
 33. The methodof claim 32, wherein the one or more modification to the cholera toxin Bsubunit variant increase the expression of the polypeptide in a plantcell.
 34. The method of claim 32, wherein the one or more modificationto the cholera toxin B subunit variant include an Asn4 to Ser mutation.35. The method of claim 34, wherein the Asn4 to Ser mutation reducesN-glycosylation.
 36. The method of claim 32, wherein the one or moremodification to the cholera toxin B subunit variant includes aC-terminal hexapeptide sequence attached for endoplasmic reticulum (ER)retention.
 37. The method of claim 36, wherein the one or moremodification to the cholera toxin B subunit variant facilitatesrecombinant production.
 38. The method of claim 32, wherein the choleratoxin B subunit variant is substantially immunologically identical to acholera toxin B subunit.
 39. The method of claim 32, wherein the woundhealing comprises mucosal wound healing.